AU745489B2 - Human neutralizing monoclonal antibodies to human immunodeficiency virus - Google Patents

Description

AUSTRAL IA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): THE SCRIPPS RESEARCH INSTITUTE Invention Title: HUMAN NEUTRALIZING MONOCLONAL ANTIBODIES TO HUMAN IMMYUNODEFICIENCY VIRUS The following statement is a full description of this invention, including the best method of performing it known to me/us: c °o.oo *o *o *o* o *oo "0;273

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HUMAN NEUTRALIZING MONOCLONAL ANTIBODIES TO HUMAN IMMUNODEFICIENCY

VIRUS

Technical Field The present invention relates generally to the field of immunology and specifically to human monoclonal antibodies which bind and neutralize human immunodeficiency virus (HIV).

Background 1. HIV Immunotherapv HIV is the focus of intense studies as it is the causative agent for acquired immunodeficiency syndrome (AIDS). Immunotherapeutic methods are one of several approaches to prevention, cure or remediation of HIV infection and HIV-induced diseases. Specifically, the use of neutralizing antibodies in passive immunotherapies is of central importance to the present invention.

Passive immunization of HIV-1 infected humans using human sera containing polyclonal antibodies immunoreactive with HIV has been reported. See for example, Jackson et al., Lancet, September 17:647-652, (1988); Karpas et al., Proc, NatL Acad. Sci.. USA, 87:7613-7616 (1990).

Numerous groups have reported the preparation of human monoclonal antibodies that neutralize HIV isolates jn vitro.

The described antibodies typically have immunospecificities for epitopes on the HIV glycoprotein gpl20 or the related external surface envelope glycoprotein gpl20 or the transmembrane glycoprotein gp41. See, for example Levy, Micro. Rev., 57:183-289 (1993); Karwowska et al., Aida Research and Human Retroviruses, 8:1099-1106 (1992); Takeda et al., J. Clin. Invest., 89:1952-1957 (1992); Tilley et.al., Aids Research and Human Retroviruses, 8:461-467 (1992); Laman et al., J. Virol., 66:1823-1831 (1992); Thali et al., J. Virol., 65:6188-6193 (1991); Ho et al., Proc. Natl. Acad.

Sci. USA, 88:8949-8952 (1991); D'Souza et al., AIDS, 5:1061- 1070 (1991); Tilley et al., Res. Virol., 142:247-259 2 (1991); Broliden et al., Immunol., 73:371-376 (1991); Matour et al., J. Immunol., 146:4325-4332 (1991); and Gorny et al., Proc. Natl. Acad. Sci., USA, 88:3238-3242 (1991).

.To date, none of the reported human monoclonal antibodies have been shown to be effective in passive immunization therapies. Further, as monoclonal antibodies, they all each react with an individual epitope on the HIV envelope glycoprotein, gpl20 or gpl60. The epitope against which an effective neutralizing antibody immunoreacts has not been identified.

There continues to be a need to develop human monoclonal antibody preparations with significant HIV neutralization activity. In addition, there is a need for TS* monoclonal antibodies immunoreactive with additional and diverse neutralizing epitopes on HIV gpl20 and gp41 in view of recent studies suggesting that gpl20 and gp41 are involved in both binding of the HIV virus to the cell as well- as in post binding events including envelope shedding and cleavage. See, for review, Levy, Micro. Rev., 57:183- 289 (1993). Additional (new) epitope specificities are required because, upon passive immunization, .the .administered patient can produce an immune response against the administered antibody, thereby inactivating 2F. the particular therapeutic antibody.

2. Human Monoclonal Antibodies Produced From Combinatorial Phacemid Libraries The use of filamentous phage display vectors, referred to as phagemids, has been repeatedly shown to allow the efficient preparation of large libraries of monoclonal antibodies having diverse and novel immunospecificities.

The technology uses a filamentous phage coat protein membrane anchor domain as a means for linking gene-product and gene during the assembly stage of filamentous phage replication, and has been used for the cloning and expression of antibodies from combinatorial..libraries..

Kang et al., Proc. Natl. Acad. Sci., USA, 88:4363-4366 tiS~t 3 (1991). Combinatorial libraries of antibodies have been produced using both the cpVIII membrane anchor (Kang et al., supra) and the cpiii membrane anchor. Barbas et al., Proc. Natl. Acad. Sci.. USA, 88:7978-7982 (1991) The diversity of a filamentous phage-based combinatorial antibody library can be increased by shuffling of the heavy and light chain genes (Kang et al., Proc. Natl. Acad. Sci.. USA, 88:11120-11123 (1991)), by altering the CDR3 regions of the cloned heavy chain genes of the library (Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461 (1992)), and by introducing random mutations into the library by error-prone polymerase chain reactions (PCR) [Gram et al., Proc. Natl. Acad. Sci., USA, 89:3576-3580 (1992)].

Filamentous phage display vectors have also been utilized to produce human monoclonal antibodies immunoreactive with hepatitis B virus (HBV) or HIV antigens. See, for example Zebedee et al., Proc. Natl.

Acad. Sci.,. USA, 89:3175-3179 (1992); and Burton et al., Proc. Natl. Acad. Sci.. USA, 88:10134-10137 (1991), respectively. None of the previously described human monoclonal antibodies produced by phagemid vectors that are immunoreactive with HIV have been shown to neutralize

HIV.

In particular, none of the previously-described human monoclonal antibodies produced by phagemid vectors are 1 capable of neutralizing a majority of the field .isolates of HIV. It is believed that certain of the antibodies described herein are particularly effective at neutralizing HIV because the antibodies immunoreact with an important antigenic determinant present on "mature" and not present on the HIV precursor protein Brief Description of the Invention In a first aspect the invention provides an isolated or substantially purified human whole. immunoglobulin molecule which is capable of immunoreacting with human immunodeficiency virus (HIV) glycoprotein gpl20 and neutralizing HIV, wherein the molecule has the capacity to reduce HIV infectivity titer in an in vitro virus 3A infectivity assay by 50% at a concentration of less than 700 nanograms (ng) of molecule per milliliter (ml) and binds mature gpl20 preferentially over HIV precursor glycoprotein In one embodiment, the human whole immunoglobulin molecule is IgGl.

In another embodiment, the human whole immunoglobulin molecule has the binding specificity of a monoclonal antibody comprising a heavy chain immunoglobulin variable region amino acid sequence shown in SEQ ID NO: 155.

S

S

V

V

V

4 Amino acid sequences which confer neutralization function to the antigen binding domain of a whole human immunoglobulin molecule and which can be used immunogenically to identify other antibodies that specifically bind and neutralize HIV are described.

The whole immunoglobulin molecules of the invention find particular utility as reagents for the diagnosis and immunotherapy of HIV-induced disease.

A major advantage of the whole immunoglobulin molecules of the invention derives from the fact that they are encoded by a human polynucleotide sequence. Thus, in vivo use of the whole immunoglobulin molecule for diagnosis and immunotherapy of HIV-induced disease greatly reduces the problems of significant host immune response to the :passively administered antibodies which is a problem commonly encountered when monoclonal antibodies of xenogeneic or chimeric derivation are utilized.

An additional major advantage of whole immunoglobulin molecules of the invention derives from the fact that they immunoreact with a unique determinant present on mature HIV glycoprotein gpl20. This class of molecules is particularly effective at neutralizing field isolates of

HIV.

A preferred whole immunoglobulin molecule has the binding specificity of an immunoglobulin comprising a heavy chain immunoglobulin variable region amino acid residue sequence selected from the group consisting of SEQ ID Nos 66, 67, 68, 70, 72, 73, 74, 75, 78 and 79.

The human whole immunoglobulin molecule may bind to a V1/V2 loop deficient-variant gpl20 substantially less than native gpl20, thereby defining an important epitope. Whole immunoglobulin molecules having these properties are particularly useful at neutralizing field isolates, and therefore provide useful information regarding the immunocompetence of an immune response in HIV-infected y D patients.

5 In another aspect, the invention provides for a screening method to determine whether HIV-infected patients contain antibodies of the class that neutralize field isolates. The method for determining immunocompetence of a human anti-human immunodeficiency virus (HIV) antibody in a sample comprises the steps of: contacting a sample believed to contain a human anti-HIV antibody with a diagnostically effective amount of the whole immunoglobulin molecule of the first aspect of the invention in a competition immunoreaction admixture containing mature gpl20 in the solid phase; maintaining the competition immunoreaction admixture under conditions sufficient for the whole immunoglobulin molecule to bind with the gpl20 in the solid phase and form a solid phase immunoreactant; and detecting the amount of the immunoreactant present in the solid phase, and thereby the immunocompetence of any human anti-HIV antibody in the sample.

A preferred whole immunoglobulin molecule has the binding specificity of an immunoglobulin comprising a light co chain immunoglobulin variable region amino acid residue sequence selected from the group consisting of SEQ ID Nos 95, 96, 97, 98, 101, 102, 103, 104, 105, 107, 110, 115, 25 118, 121, 122, 124 and 132.

In a second aspect the invention provides a polynucleotide sequence encoding a heavy or light chain immunoglobulin variable region amino acid residue sequence portion of a whole immunoglobulin molecule of the first aspect of the invention.

In a third aspect the invention provides DNA expression vectors containing the polynucleotide of the second aspect of the invention.

In a fourth aspect the invention provides host cells containing the vectors of the third aspect of the ?ALI invention.

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6 In a fifth aspect the invention provides a method of detecting human immunodeficiency virus (HIV) comprising contacting a sample suspected of containing HIV with a diagnostically effective amount of the whole immunoglobulin molecule of the first aspect of the invention, and determining whether the whole immunoglobulin molecule immunoreacts with the sample. The method can be practiced in vitro or in vivo, and may include a variety of methods for determining the presence of an immunoreaction product.

In a sixth aspect the invention provides a method for providing passive immunotherapy to human immunodeficiency virus (HIV) disease in a human, comprising administering to the human an immunotherapeutically effective amount of the whole immunoglobulin molecule of the first aspect of the invention. The administration can be provided ':prophylactically, and by a parenteral administration. In a seventh aspect, the invention provides pharmaceutical compositions containing one or more of the whole immunoglobulin molecules of the first aspect of the invention for use in the therapeutic methods of the invention.

Brief Description of the Drawings In the drawings forming a portion of this -disclosure: Figure 1 illustrates the sequence of the double-stranded synthetic DNA inserted into Lambda Zap to produce a Lambda Hc2 expression vector. The preparation of the double-stranded synthetic DNA insert is described in Example la2) The various features required for this vector to express the V.-coding DNA homologs include the Shine-Dalgarno ribosome binding site, a leader sequence to direct the expressed protein to the periplasm as described by Mouva et al., J. Biol. Chem., 255:27, 1980, and various restriction enzyme sites used to operatively link the V.

homologs to the expression vector. The V. expression vector sequence also contains a short nucleic acid X sequence that codes for amino acids typically found in Vt variable regions heavy chain backbone). This V.

backbone is just upstream and in the proper reading as the VH DNA homologs that are operatively linked into the Xho I and Spe I cloning sites. The sequences of the top and bottom strands of the double-stranded synthetic DNA insert are listed respectively in SEQ ID NO 1 and SEQ ID NO 2.

The ten amino acid sequence comprising the decapeptide tag is listed in SEQ ID NO 5. The synthetic DNA insert is directionally ligated into Lambda Zap II digested with the restriction enzymes Not 1 and Xho I to form Lambda Hc2 expression vector.

Figure 2 illustrates the major features of the bacterial expression vector Lambda Hc2 expression vector) The orientation of the insert in Lambda Zap II go e C e°

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8 is shown. The VH DNA homologs are inserted into the Xho I and Spe I cloning sites. The read through transcription produces the decapeptide epitope (tag) that is located just 3' of the cloning site. The amino acid residue sequence of the decapeptide tag and the Pel B leader sequence/spacer are respectively listed in SEQ ID NO 5 and 6.

Figure 3 illustrates the sequence of the doublestranded synthetic DNA inserted into Lambda Zap to produce a Lambda Lc2 expression vector. The various features required for this vector to express the VL-coding DNA homologs are described in Figure 1. The VL-coding DNA homologs are operatively linked into the Lc2 sequence at the sac I and Xho I restriction sites. The sequences of the top and bottom strands of the double-stranded .synthetic DNA insert are listed respectively in SEQ ID NO 3 and SEQ ID NO 4. The synthetic DNA insert is directionally ligated into Lambda Zap II digested with the restriction enzymes Sac I and Not I to form Lambda Lc2 •20 ,expression vector.

Figure 4 illustrates the major features of the- bacterial expression vector Lc2 (VL expression vector).

The synthetic DNA sequence from Figure 3 is shown at the top along with the LacZ promoter from Lambda Zap II. The :l orientation of the insert in Lambda Zap II is shown. The

V

1 DNA homologs are inserted into the Sac I and Xho I cloning sites. The amino acid residue sequence of the Pel B leader sequence/spacer is listed in SEQ ID NO 7. Figure 5 illustrates the dicistronic expression vector, pComb, in the form of a phagemid expression vector.

Figure 6 illustrates the neutralization of HIV-1 by recombinant Fabs. The same supernate preparations were used in p24 and syncytia assays. The figures indicate neutralization titers. Refer to Example 3 for details of the assay procedures and discussion of the results. The ELISA titers and Fab concentrations were determined as described in Example 2b.

~~-S~vS~zi2. 4> 9 Figure 7 illustrates the relative affinities of Fab fragments for gpl20 (IIIB) as illustrated by inhibition ELISA performed as described in Example 2b6). Fabs 27, 6, 29, 2 and 3 are all prototype members of the different groups discussed in Example 9a. Loop 2 is an Fab fragment selected from the same library as the other Fabs but which recognizes the V3 loop. The data is plotted as the percentage of maximum binding on the Y-axis against increasing concentrations (10 11. M to 10,7 M) of soluble gpl20 on the X-axis.

Figure 8 illustrates the soluble CD4 competition with Fab fragments for gpl20 (IIIB) P4D10 and loop2 are controls. P4D10 is a mouse monoclonal antibody reacting with the V3 loop of gpl20 (IIIB). The data, discussed in 15 Example 2b6), is plotted as described in Figure 7.

Figure 9 illustrates the neutralization of HIV by purified Fabs prepared as described in Example 3. The results shown are derived from the syncytia assay using the MN strain. The data is plotted as percent of 20 inhibition of binding-on the Y-axis against increasing Fab concentrations (0.1 to greater than micrograms/milliliter (Ag/ml)] on the X-axis.

Figures 10A and 10B illustrate the amino acid residue sequences of variable heavy (V

H

domains of Fabs binding to gpl20. Seven distinct groups have been identified as described in Example 9a based on sequence homology.

Identity with the first sequence in a group is indicated by dots. The Fab clone names are indicated in the left hand column. The corresponding SEQ ID Nos are indicated in the'right hand column. The sequenced regions from right to left are framework region 1 (FRI), complementary determining region 1 (CDR1), framework region 2 (FR2), complementary determining region 2 (CDR2) framework region 3 (FR3), complementary determining region 3 (CDR3), and framework region 4 (FR4) The five amino-terminal residue sequence beginning with LEQ arises from the VHia while the 5 amino-terminal residue sequence beginning with LEE arises from the VH3a primers. The bil and b29 ;zrc,-c ~-V2Zq-E4t~#At~-<t- 10 sequences are very similar to the b3 group and could be argued to be intraclonal variants within that group; they are placed in their own group because of differences at the V-D and D-J interface.

Figures 11A and 11B illustrate the amino acid residue sequences of variable light (VL) domains of Fabs binding to Refer to Figures 10A and 10B for the description of the figure and to Example 9b for analysis of the sequences.

1. 10 Figures 12A and 12B illustrate the amino acid residue sequences of VL domains from Fabs binding to gpl20 and generated by shuffling the heavy chain from clone b12 against a library of light chains (H12-LCn Fabs) as described in Example 10. Note that the new VL sequences have designated clone numbers that do not relate. to those numbers from the original library. The unique sequences are listed in the Sequence Listing from SEQ ID NO 114 to 122. The new VL domain sequences are compared to that of the original clone b12 VL sequence.

Figures 13A and 13B illustrate the amino acid residue sequences of VH domains from Fabs binding to gpl20 and generated by shuffling the light chain from clone b12 against a library of heavy chains (L12-HCn Fabs) as described in Example 10. Note that the new VH sequences have designated clone numbers that do not relate to those numbers from the original library. The unique sequences are listed in the Sequence Listing from SEQ ID NO 123 to 132. The new V H domain sequences are compared to that of the original clone b12 V. sequence.

Figures 14A and 14B illustrate plasmid maps of the heavy (pTAC01H) and light chain (pTC01) replicon-compatible chain-shuffling vectors, respectively.

Both plasmids are very similar in the section containing the promoter and the cloning site. Abbreviations: tacPO, tac promoter/operon; 5.histidine amino acid residue .tag flIG, intergenic region of fl-phage; stu, stuffer fragment ready for.in-frame replacement by light and heavy chain, respectively; cat, chloramphenicol Z.z. sm4a,.~.

11 transferase gene; bla, b-lactamase gene; ori, origin of replication. The map is drawn approximately to scale.

Figures 15A and 15B illustrate the nucleotide sequences of the binary shuffling vectors. The construction and use of the vectors is described in Example 11. In Figure the double-stranded nucleotide. sequence of the multiple cloning site in light chain vector, pTC01, is shown. The sequences of the top and bottom nucleotide base strands are listed respectively in SEQ ID NO 8 and SEQ ID NO 9.

The amino acid residue sequence comprising the pelB leader ending in the Sac I restriction site is listed in SEQ ID NO 10. In Figure 15B, the nucleotide sequence of the multiple cloning site in heavy chain vector, pTAC01H, is shown. The sequences of the top and bottom nucleotide base strands are listed respectively in SEQ ID NO 11 and SEQ ID NO 12. The amino acid residue sequence comprising the pelB leader ending in the Xho I restriction site is listed as SEQ ID NO 13. The amino acid residue sequence comprising the histidine tail is listed in SEQ ID NO 14.

20 Relevant restriction sites are underlined. tac promoter .and ribosome binding site .(rbs) are indicated by boxes.

Figure 16 -illustrates the complete set of directed crosses between heavy and light chains of all Fab 2 fragments isolated from the original library by panning with gpl60 (IIIB) (bl-b27), gpl20 (IIIB) (B8-B35), (SF2) (s4-s8), and the'loop peptide (p35) assayed by ELISA against IIIB gpl20 as described in Example 11. Heavy chains are listed horizontally and light chains are listed vertically. Clones are sorted according to the grouping established in Example 9. Different groups are separated by horizontal and vertical lines. A at the intersection of a particular heavy chain and light chain signifies a clear negative (a signal of 3 times background or less) for that particular cross, a shows a clear positive comparable to the original heavy and light chain combination, and a denotes an intermediate value in the ELISA. the HCp35/ LCp35 combination is negative when gpl20 (IIIB) is used, but positive when assayed with 12 (IIIB). Identical chains carry the same identifier (either or Y) Figure 17 illustrates the affinity of antibody-antigen interaction for b12 heavy chain crosses with light chains from all pannings analyzed by competitive ELISA using soluble IIIB gpl20 as competing antigen as described in Example 10. The data is plotted as the percentage of maximum-binding on the Y-axis against increasing concentrations of soluble gpl20 (IIIB) (10- 12 M to 10- 7

M)

on the X-axis.

Figures 18A and 18B illustrate the amino acid residue sequences of variable heavy domains of Fabs binding to gp41. The Fab clone names are indicated in the left hand .column. The heavy chain sequences of the five Fabs individually designated DL 41 19, DO 41 11, GL 41 1, MT 41.

;12 and SS 41 8 have been assigned the respective SEQ ID Nos 142, 143, 144, 145 and 146. The sequenced regions from right to left are framework region 1 (FR1), complementary determining region 1 (CDR1), framework 2 0 %region 2 (FR2), complementary determining region 2 (CDR2), framework region 3 (FR3), complementary determining region l 3 (CDR3), and framework region 4 (FR4) -Figures 19A and 19B illustrate the amino acid residue sequences of variable light (VL) domains of Fabs binding to gp41. Refer to Figures 18A and 18B for the description of the figure. The light chain sequences of the five Fabs •I individually designated DL 41 19, DO 41 11, GL 41 1, MT 41 12 and SS 41 8 have been assigned the respective SEQ ID NOs 147, 148, 149, 150 and 151.

Figure 20 illustrates the relative binding affinities of b3, b6, and b12 for the total envelope glycoproteins and for the gpl20 glycoprotein (gpl20) expressed on the surface of COS-1 cells as determined by immunoprecipitation and described in Example 6. The signal on the autoradiogram represents the relative amount of envelope glycoproteins bound with increasing concentrations of Fab (0-150 Ag/ml).

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13 Figure 21 illustrates the neutralization of HIV-1 by b12 IgG-l as assessed using PHA-stimulated PBMCs as indicator cells and determination of extracellular p24 as the reporter assay. Refer to Example 5d for details of the assay procedures and discussion of the results. The designation, location, and disease status of the virus donors were as follows: X, VS (New York, acute), T, N70-2 (New Orleans, asymptomatic), A, AC (San Diego, AIDS), 0, LS (Los Angeles, AIDS), D, NYC-A (New York, unknown), v, WM (Los Angeles, AIDS), A, RA (New York, acute), o, JP (New York, acute). The molecularly cloned HIV-1 virus JR- CSF and HIV-1 isolate JR-FL were also assayed for neutralization. The data is plotted as neutralization on the Y-axis against increasing concentrations of b12 15 IgGI (0-25 Ag/ml) on the X-axis.

Figure 22 illustrates the reactivity of b12 IgGl with a .panel of international isolates of HIV-1 as described in Example 8. Reactivity was determined with gpl20 isolated from the HIV-1 samples in ELISA with the b12 IgGl as 20 described in Example 8. Data is plotted as b12 IgGl *e reactivity on the X-axis against clades A-F on the Y-axis.

Country names indicate where the HIV-1 virus was originally isolated. The numbers in parenthesis refer to the number of viruses of each clade examined. Reactivity is designated as strong or moderate (E Figure 23 illustrates the neutralization of the HXBc2 molecular clone of HIV-1 LAI by purified Fabs and a monoclonal antibody 110.4 (Mab 110.4) in an envelope complementation assay as described in Example 3c.

Neutralization of HXBc2 infectivity is expressed as a decrease in residual CAT activity. The data is plotted as residual CAT activity on the Y-axis and increasing concentrations of Fab and MAb (0.1-20 Ag/ml) on the Xaxis.

Figure 24 illustrates the pSG-5 mammalian expression vector as described in Examples 4a and 4b. Transcription of the heavy or light chain gene when inserted in the EcoRI site is under the control of the SV40 early 14 promoter. Transcriptional termination is signaled by the polyadenylation signal sequence downstream of the heavy chain sequence. The M13 intergenic region allows for the production of single-stranded DNA for nucleotide sequence determination. The ampR gene is for selection of tHe vector in bacterial cells.

Figures 25A and 25B illustrate the nucleotide and amino acid residue sequences of the b12 light chain gene in the pSG-5 mammalian expression vector described in Example 4b.

eg.

:0 The b12 light chain has been modified for expression in mammalian cells as described in Example 4b.

.Figure 26 illustrates pEe6HC BM12, the pEE6 mammalian expression vector with the b12 IgGl heavy chain gene that has been modified for antibody expression in mammalian cells as described in Example 4d. The VH was originally derived from the Fab b12 and has the same binding specificity as the Fab b12. The pEE6 vector has a human CMV promoter for expression of the heavy chain, a polyadenylation signal for termination of transcription, and an ampicillin gene for selection in bacteria.

Figures 27A through 27E illustrate the nucleotide *sequences of the b12 heavy chain VH and constant regions in the pEe6HC BM12 mammalian expression vector as described Example 4d. The amino acid residue sequence of the b12 heavy chain VH is given. The b12 VH has been modified for expression in mammalian cells as described in Example 4d.

Figure 28 illustrates pEel2 Combo BMl2, the pEEl2 mammalian expression vector with b12 IgGi heavy and light chain genes that have been modified for antibody expression in mammalian cells as described in example 4f.

The VH and light chain were originally derived from the Fab b12 and have the same binding specificity as the Fab.

b12. The pEEl2 vector has-a human CMV promoter for expression of the light chain, a polylinker to provide cloning sites, and a polyadenylation signal for termination of transcription. The vector also contains the GS selectable marker gene whose expression is

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15 controlled an SV40 early promoter at the 5' end of the GS gene, an intron, and a polyadenylation signal at the 3' end of the GS gene. A heavy chain cassette comprising the HCMV promoter, enhancer elements, heavy chain gene, and polyadenylation signal were removed from the pEE6 vector and inserted into the pEE12 vector to generate the cofbinatorial construct containing both the bl2 light and heavy chain genes.

Figure 29A through 29R illustrates the nucleotide sequence of the pEEl2 mammalian expression vector and the b12 IgGI heavy and light chain genes, pEel2 Combo BM 12, o as described in Example 4f. The VH and light chain genes *°ooo.

have been modified for expression in mammalian cells as described in Example 4.

Detailed Description of the Invention A. Definitions Amino Acid Residue: An amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its n e :20 peptide linkages. The amino acid residues described g* herein are preferably in the isomeric form. However, residues in the isomeric form can be substituted for H 66. any L-amino acid residue, as long as the desired functional property is retained by the polypeptide. NH 2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature (described in J. Biol.

Chem., 243:3552-59 (1969) and adopted at 37 CFR abbreviations for amino acid residues are shown in the following Table of Correspondence: TABLE OF CORRESPONDENCE SYMBOL AMINO ACID 1-Letter 3-Letter Y Tyr tyrosine G Gly glycine S 16

O

15 F Phe phenylalanine M Met methionine A Ala alanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine V Val valine P Pro proline K Lys lysine H His histidine Q Gin glutamine E Glu glutamic acid Z Glx Glu and/or Gin W .Trp tryptophan R Arg arginine D Asp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine X Xaa Unknown or other It should be noted that all amino acid residue sequences represented herein by formulae have.a left- toright orientation in the conventional direction of amino terminus to carboxy terminus. In addition, the phrase "amino acid residue" is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those listed in 37 CFR 1.822(b)(4), and incorporated herein by reference.

Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or a covalent bond to an aminoterminal group such as NH 2 or acetyl or to a carboxyterminal group such as COOH.

Recombinant DNA (rDNA) molecule: A DNA molecule produced by operatively..linking two DNA segments. Thus, a recombinant DNA molecule is a hybrid DNA molecule ;ii; 17 comprising at least two nucleotide sequences not normally found together in nature. RDNA'S not having a common biological origin, evolutionarily different, are said to be "heterologous".

Vector: A RDNA molecule capable of autonomous replication in a cell and to which a DNA segment, e.g., gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as "expression vectors". Particularly important vectors allow cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.

Receptor: A receptor is a molecule, such as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to another molecule.

.Antibody: The term antibody in its various grammatical forms is used herein to refer to whole immunoglobulin molecules.

Antibody Combinina Site: An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds "(immiunreacts with) an antigen. The term immunoreact in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

Monoclonal Antibody: A monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contain only one species of antibody combining site capable of immunoreacting with. a 18 particular epitope. A monoclonal antibody thus typically displays a single binding affinity for any epitope with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different epitope, a bispecific monoclonal antibody.

Although historically a monoclonal antibody was produced by immortalization of a clonally pure immunoglobulin secreting cell line, a monoclonally pure population of 1.0 antibody molecules can also be prepared by the methods of the present invention.

Fusion Polypeptide: A polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous 15 polypeptide. The two polypeptides linked in a fusion C e polypeptide are typically derived from two independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature.

Upstream: In the direction opposite to the direction of DNA transcription, and therefore going from.

5' to 3' on the non-coding strand, or 3' to 5' on the mRNA.

Downstream: Further along a DNA sequence in the direction of sequence transcription or read out, that is traveling in a to 5'-direction along the non-coding strand of the DNA or to 3 1 -direction along the RNA transcript.

Cistron: Sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence and including upstream and downstream DNA expression control elements.

Leader Polvoeptide: A short length of amino acid sequence at the amino end of a polypeptide, which carries or directs the polypeptide through the inner membrane and so ensures its eventual secretion into the periplasmic space and perhaps beyond. The leader sequence peptide is commonly removed before the polypeptide becomes active.

Reading Frame: Particular sequence of contiguous 19 nucleotide triplets (codons) employed in translation. The reading frame depends on the location of the translation initiation codon.

B. Whole Immunoglobulin Molecules The present invention relates to human whole immunoglobulin molecules, (referred to below as "human monoclonal antibodies") which are specific for, and neutralize human immunodeficiency virus (HIV). Human monoclonal antibodies are disclosed which are capable of binding epitopic polypeptide sequences in glycoprotein of HIV. Human monoclonal antibodies capable of binding epitopic polypeptide sequences in glycoprotein gp 41 of HIV are disclosed. Also disclosed is an antibody having a specified amino acid sequence, which "sequence confers the ability to bind a specific epitope and to neutralize HIV when the virus is bound by these antibodies. A human monoclonal antibody with a claimed *specificity, and like human monoclonal antibodies with like specificity, are useful in the diagnosis and immunotherapy of HIV-induced disease.

The term "HIV-induced disease" means any disease caused, directly or indirectly, by HIV. An example of a HIV-induced disease is acquired autoimmunodeficiency syndrome (AIDS), and any of the numerous conditions associated generally with AIDS which are caused by HIV infection.

Thus, the present invention is directed to human monoclonal antibodies which are reactive with a HIV neutralization site and cell lines which produce such antibodies. The isolation of cell lines producing monoclonal antibodies of the invention is described in great detail further herein, and can be accomplished using the phagemid vector library methods described herein, and using routine screening techniques which permit determination of the elementary imnunoreaction and neutralization patterns of the monoclonal antibody of interest. Thus, if a human 20 monoclonal antibody being tested binds and neutralizes HIV in a manner similar to a human monoclonal antibody produced by the cell lines of the invention then the tested antibody is considered equivalent to 'an antibody of the..invention.

It is also possible to determine, without undue experimentation, if a human monoclonal antibody has the same equivalent) specificity as a human monoclonal antibody of this invention by ascertaining whether the former prevents the latter from binding to HIV. If the human monoclonal antibody being tested competes with the :human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of *the invention in standard competition assays for binding to a solid phase antigen, for example to gp126, then it is likely that the two monoclonal antibodies bind to the same, or a closely related, epitope.

.'Still another way to determine whether.'a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with HIV with which it is normally reactive, and then add the human ::.monoclonal antibody being..tested to determine if 'the human monoclonal antibody 'being testedis inhibited in its 'ability to bind HIV. If the human monoclonal antibody being teste'd is inhibited then, in all likelihood, it has the same, or functionally equivalent,. epitopic specificity as the monoclonal antibody of the-invention. Screening of human monoclonal antibodies of the invention, can be also carried out utilizing'HIV neutralization assays and determining whether the monoclonal antibody neutralizes

HIV.

The ability to neutralize IV at one or more stages of virus infection is a desirable quality of a human monoclonal antibody 'of the present invention. Virus neutralization can be measured by a variety of in vitro and in vivo methodologies.- Exemplary methods described herein for determining the capacity for neutralization are 21 the in vitro assays that measure inhibition of HIV-induced syncytia formation, plaque assays and assays that measure the inhibition of output of core p24 antigen from a cell infected with HIV.

As shown herein, the immunospecificity of a human monoclonal antibody of this invention can be directed to epitopes that are shared across serotypes and/or strains of HIV, or can be specific for a single strain of HIV, depending upon the epitope. Thus, a preferred human monoclonal antibody can immunoreact with HIV-1, HIV-2, or both, and can immunoreact with one or more of the HIV-1 strains IIIB, MN, RF, SF-2, Z2, Z6, CDC4, ELI and the like strains. In addition, a preferred human monoclonal antibody can immunoreact and neutralize a majority of field isolates of HIV, as described further herein.

The immunospecificity of an antibody, its HIVneutralizing capacity, and the attendant affinity the antibody exhibits for the epitope, are defined by the epitope with which the antibody immunoreacts. The epitope specificity is defined at least in part by the amino acid residue sequence of the variable region of the heavy chain of the immunoglobulin the antibody, and in part by the light chain variable region amino acid residue sequence.

Preferred human monoclonal antibodies immunoreact with the CD4 binding site of glycoprotein Also disclosed is an antibody having a specified e' amino acid sequence, which sequence confers the ability to bind a specific unique neutralizing epitope and to neutralize HIV when the virus is bound by these antibodies.

As described above, human monoclonal antibodies which have the binding specificity of a monoclonal antibody comprising a heavy chain immunoglobulin variable region amino acid residue sequence selected from the group of sequences consisting of SEQ ID NOs 66, 67, 68, 70, 72, 73, 74, 75, 78 and 79, and conservative substitutions thereof are exemplary of monoclonal antibodies within the broad description of the invention.

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t- 22 Also described above, hunan monoclonal antibodies which have the binding specificity of a monoclonal antibody having a light chain immunoglobulin variable region amino acid residue sequence selected from the group of sequences consisting of SEQ ID NOs 95, 96, 97, 98, 101, 10-2, 103, 104, 105, 107, 110, 115, 118, 121, 122, 124 and 132, and conservative substitutions thereof are exemplary of monoclonal antibodies within the broad description of the invention.

Monoclonal antibodies of this invention exhibit a potent capacity to neutralize HIV. The capacity to neutralize HIV is expressed as a concentration of antibody molecules required to reduce the infectivity titer of a suspension of HIV when assayed in an typical in vitro infectivity assay, such as is described herein. A monoclonal antibody of the first aspect of the invention has the capacity to reduce HIV infectivity titer in an in vitro virus infectivity assay by 50% at a concentration of less than 700 nanograms (ng) of antibody per milliliter (ml) of culture medium in the assay, and preferably reduces infectivity titers 50% at a concentration of less than 300 ng/ml, and more preferably at concentrations less than about 10 ng/ml.

Exemplary and preferred monoclonal antibodies described herein are effective at 3-700 ng/ml, and therefore are particularly well suited for inhibiting HIV in vitro and in vivo.

Human monoclonal antibodies of this invention immunoreact with gpl20 in its "mature" form, which form is to be distinguished from antigenic determinants present on the HIV envelope precursor glycoprotein designated gpl60. gpl60 is processed during virus biogenesis by cleavage into two polypeptides, gp41 and gpl20. "Mature" gpl20 refers to the processed protein that is found in mature HIV virus particles, and can be detected on the surface of HIV-infected cells.

Thus, whole immunoglobulin molecule of the first aspect of the invention binds mature gpl20 preferentially over HIV precursor glycoprotein gpl60. By "binds uLJ4mv preferentially" is meant that the whole immunoglobulin u molecule immunoreacts with (binds) substantially ''li XIF~...ii~D 23 more mature gpl20 than gpl60 in an immunoreaction admixture. Substantially more typically indicates that at least greater than 50 of the total mass of immunoprecipitated material is gpl20, and preferably indicates that at least greater than 75 more preferably of the immunoprecipitated material is gpl2 0 Methods for determining immunoreaction of a subject antibody with gpl20 or gpl60 are well known in the art, and the invention need not be so limited. However, preferred methods for determining the relative amounts of envelope glycoprotein antigens are described in the Examples, and include radio-immunoprecipitation (RIP) of cell-surface labeled HIV-infected cells, followed by molecular weight, analysis of the labeled products by polyacrylamide gel electrophoresis

(PAGE)

A preferred human monoclonal antibody also has the ability to immunoreact with nat-ive gpl 2 0 and comparatively bind substantially less of a variant gpl 2 0 produced by recombinant.DNA methods in which the V1 and V2 loops have S" been deleted. The variant gpl20, also referred to a V1/V2 loop deficient-variant gpl 2 0, is described in the Examples, and is seen to bind substantially less of.

antibody, b12, in comparison to native The term "native gpl 2 0" refers to a mature gpl20 protein having a normal amino acid residue sequence instead of a variant protein having selected amino acid residue substitutions or deletions, such as the V1/V2 loop deficient-variant in which the Vl and V2 loops were deleted. This preferential binding with native gpl 2 0 compared to the V1/V2 loop deficient-variant identifies an important epitope defined by a preferred antibody of this invention. Antibodies having this binding epitope are particularly effective at neutralizing a majority of field isolates of HIV, as described herein.

The ability to bind "substantially less" Vi/V2 loop deficient-variant gpl 2 0 than native gpl 2 0 can be readily measured using various immunoreaction detection methods, although the assay methods described in Example 24 are. particularly preferred. In preferred embodiments, substantially less binding to Vl/V2 loop deficient-variant gp12O compared to native gp12O is indicated when the comparison is conducted as described as in Example Sc, and the native gpl2Q exhibits a ratio value deviating from the mean of greater than 2.0 and the variant exhibits a ratio value deviating from the mean of less than A particularly preferred human monoclonal antibody of this invention also has the capacity to neutralize a majority of field isolates as disclosed herein. As is well understood, the field clinically isolated) strains of HI-V are typically different to some degree antigenically from laboratory strains. Therefor, it is well understood that useful neutralizing antibodies must immunoreact with, and be neutr alizing against, field isolates of HIV. Preferably, the useful antibody neutralized a large percentage of field isolates, thereby increasing its effectiveness when new strains are encountered.

The Examples demonstrate that the human monoclonal antibody b12 has the ability to neutralize a majority of the field isolates tested. By majority is meant that in a representative and diverse collection of_ field isolates, the 'antibody is capable of neutralizing at least 50 of the strains, and preferably at least 75 k of the strains tested. In this context, "neutralizing"i means an effect :of reducing the HIV infectivity titre in an in vitro virusinfectivity assay as described herein at the antibody concentrations described.

Thus in a preferred embodiment of the first aspect of *the invention, the invention provides a whole immunoglobulin molecule capable of immunoreacting with and neutralizing a first preselected human immunodeficiency virus (HIV), such as the laboratory isolate MN or 1112, that is further capable of immunoreacting with and neutralizing one or more other second) strains of HIV, particularly field strains. In this embodiment, supported by the teachings of the Examples, the whole immunoglobulin molecule has the capacity to reduce HIV infectivity titer in an in 25 vitro virus infectivity assay of the first HIV strain by__ at a concentration of at least less than (ng) of antibody per milliliter (ml) and has the capacity to reduce HIV infectivity titer of a second field strain of HIV in the same in vitro virus infectivity assay by. 50% at a concentration of less than about 700 nanograms (ng) of antibody per milliliter (ml) In more preferred embodiments and depending upon the particular HIV strain, the capacity to reduce second field strain infectivity titers by 50*% can be exhibited at lower antibody concentrations, such as below 300 ng/ml.

An antibody having the binding specificity of the b12 monoclonal antibody described herein is exemplary of an antibody within the broad description of the invention.

The amino acid residue sequence of the heavy chain variable region of b12 is shown in SEQ ID No 66, and the light chain variable region sequence of b12 is shown in SEQ ID NO 97.

Human antibodies having the binding specificity of the *immunoglobulin heavy and light chain polypeptides produced by ATCC 69079 are also equally exemplary of an antibody within the broad description of the invention.

Human monoclonal antibodies which immunoreact with the CD4 binding site of glycoprotein gp4l are described herein.

Such antibodies include a human monoclonal antibody which has the binding specificity of a monoclonal antibody comprising a heavy chain immunoglobulin variable region amino acid residue sequence selected from the group of sequences consisting of SEQ ID Nos 142, 143, 144, 145 and 146 and conservation substitutions thereof.

Further antibodies include a human monoclonal antibody which has the gp4l binding specificity of a monoclonal antibody having a light chain inmunoglobulin variable region amino acid residue sequence selected from the group of sequences consisting of SEQ ID NOs 147, 148, 149,150, and 151 and conservative substitutions thereof.

26 As shown by the present teachings and using the combinatorial library shuffling and screening methods, one can identify new heavy and light chain pairs that function as a HIV-neutralizing monoclonal antibody. In particular, -one can shuffle a known heavy chain, derived from an HIV-neutralizing human monoclonal antibody, with a library of light chains to identify new'H:L pairs that.

form a functional antibody according to the present invention. Similarly, one can shuffle a known light chain, derived from an HIV-neutralizing human monoclonal antibody, with a library of heavy chains to identify new H:L pairs that form a functional antibody according to the present invention.

Human monoclonal antibodies which have the immunoreaction (binding) specificity of a monoclonal antibody having heavy and light chain immunoglobulin variable region amino acid residue sequences in pairs selected from the group consisting of SEQ ID NOs 66:95, 67:96, 72:102, 66:97, oee 73:107, 74:103,70:101, 68:98, 75:104, 72:105, 78:110, 66:118, 66:122, 66:121,66:115,79:124, 79 :132 and 66:98, and conservative substitutions thereof are exemplary of monoclonal antibodies within the broad description of the invention. The designation of two SEQ ID NOs with a colon, 66:95, is to 9 connote a H:L pair formed by the heavy and light chain, respectively, amino acid residue sequences shown in SEQ ID NO 66 and SEQ ID NO 95, respectively.

aoo o a Human monoclonal antibodies having the gp4l Cimmunoreaction (binding) specificity of a monoclonal antibody having heavy and light chain immunoglobulin variable region amino acid residue sequences in pairs (H:L) selected from the group consisting of SEQ ID NOs 142:147, 143:148, 144:149, 145:150, and 146:151, and conservative substitutions thereof are described.

Human monoclonal antibodies having the binding specificity of the monoclonal antibody produced by the E. coli microorganisms deposited with the ATCC, as described further 27 herein are exemplary of monoclonal antibodies within the broad description of the invention. Included amongst these are human monoclonal antibodies having the binding specificity of the monoclonal antibodies produced by the E. coli microorganisms designated ATCC 69078, 69079 and 69080. By "having the binding specificity" is meant equivalent monoclonal antibodies which exhibit the same or similar immunoreaction and neutralization properties, and which compete for binding to an HIV antigen for example, human monoclonal antibodies produced by ATCC 69078, 69079 and 69080.

The term "conservative variation" as used herein denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of *e conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar e g residue for another, such as the substitution of arginine o for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. The term "conservative variation" also includes the'use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies having the substituted polypeptide also neutralize HIV. Analogously, in the second aspect the invention provides polynucleotides which encode the above noted heavy and/or light chain polypeptides and to polynucleotide sequences which are complementary to these polynucleotide sequences.

Complementary polynucleotide sequences include those sequences which hybridize to the polynucleotide sequences of the invention under stringent hybridization conditions.

By using the human monoclonal antibodies of the invention, it is now possible to produce anti-idiotypic antibodies which can be used to screen human monoclonal antibodies to identify whether the antibody has the same binding specificity as a human monoclonal antibody of the invention and also used for active immunization (Herlyn et al., Science, 232:100 (1986)). Such anti-idiotypic Santibodies can be produced using well-known hybridoma -i: ;~ii1.7 ;I 28 techniques (Kohler et al., Nature, 256:495 (1975)). An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the human monoclonal antibody produced by the cell line of interest. These determinants are located in the hypervariable region of the antibody. It is this region which binds to a given epitope and, thus, is responsible for the specificity of the antibody. An anti-idiotypic antibody can be prepared by-immunizing an animal with the monoclonal antibody of interest. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an antibody to these idiotypic determinants.

By using the anti-idiotypic antibodies of the immunized animal, which are specific for the human monoclonal antibody of the invention produced by a cell line which was used to immunize the second animal, it is now possible to identify other clones with the same idiotype as the antibody of the hybridoma used for immunization.

Idiotypic identity between human monoclonal antibodies of two cell lines demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant. Thus, by using antiidiotypic antibodies, it is possible to identify other hybridomas expressing monoclonal antibodies having the same epitopic specificity.

It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope.* For example, an anti-idiotypic monoclonal antibody.made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody. Thus, the anti-idiotypic monoclonal antibody .can be used for immunization, since the anti-idiotype monoclonal antibody binding domain effectively acts as an antigen.

29 The invention provides a whole immunoglobulin molecule comprising a Fab fragment derived from a human monoclonal antibody of this invention and the fragment crystallizable (Fc) domain of a human immunoglobulin molecule. The entire complete) immunoglobulin molecule comprising a Fab fragment with the Fc domain may afford therapeutic and diagnostic advantages, and can be any of the several Ig species depending upon the ultimate use,'including IgG, IgA, IgD, IgE, IgM, and isotypes thereof. The immunoglobulin molecule would be capable of effector functions associated with the Fc domain when used in passive immunotherapy. These effector functions include antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDCC) which promote the death of the cell to which the immunoglobulin molecule is specifically bound. The effector functions :may therefore be desirable in therapeutic applications.

Diagnostic assays include the ability to detect the presence of the immunoglobulin molecule. These assays rely on the cross-linking of red cells or beads in agglutinations, the activation of complement in plaque assays, or the antigenic properties of the Fc region of the heavy chain as detected by secondary antibodies in ELISA or RIA procedures to detect the presence of the immunoglobulin molecule. Such diagnostic assays can only be performed with the entire immunoglobulin molecule. The isolation of the immunoglobulin molecule is also facilitated by the presence of the Fc domain in that commonly used methods of immunoglobulin purification are based upon interaction of reagents with the Fc domain.

The preparation of a Fab fragment with the Fc domain is generally known in the immunological arts and can be accomplished by a variety of methods. A preferred method l.r; 7 30 of producing a Fab fragment with the Fc domain is described herein.

The immunoglobulin IgGI human antibody described herein that is comprised of the b12 antibody Fab fragment and human Fc domain derived from an IgGI subtype, designated b12 IgGI is exemplary of an immunoglobulin molecule comprising a Fab fragment derived from a human monoclonal antibody and the Fc domain of a human immunoglobulin molecule. The structure and preparation of this preferred human monoclonal antibody is described herein, and is prepared using the recombinant DNA expression vector pEEl2. The complete nucleotide sequence of the vector for expression the complete heavy and light chains in the form of b12 IgGI is shown in Figure 27 and also in SEQ ID NOs 156 and 170.

."Accordingly, the amino acid"residue and nucleotide *sequences, respectively, for a preferred complete heavy chain are shown in SEQ ID NOs 155 and 154, respectively, and for a preferred light chain are shown in SEQ ID NOs •153, and 152, respectively. The nucleotide sequences for preferred heavy and light chains are also shown in SEQ ID NOs 169 and 168, respectively.

C. Immunotherapeutic Methods and Compositions The human monoclonal antibodies can also be used immunotherapeutically for HIV disease. The term "immunotherapeutically" or "immunotherapy" as used herein in conjunction with the monoclonal antibodies of the invention denotes both prophylactic as well as therapeutic.

administration. Thus, the monoclonal antibodies can be administered to high-risk patients in order to lessen, the likelihood and/or severity of HIV-induced disease, administered to patients already evidencing active HIV infection, or administered to patients at risk of HIV infection.

1. Therapeutic Compositions The present invention therefore contemplates therapeutic compositions useful for practicing the therapeutic methods described herein.

Therapeutic compositions of the present invention contain

S

31 a physiologically tolerable carrier together with at least one. species of human monoclonal antibody as described herein,,dissolved or dispersed therein as an active ingredient. In a preferred embodiment, the therapeutic co mposition is not immunogenic when administered to a human patient for therapeutic purposes, unless that purpose is to induce an immune response, as described elsewhere herein.

As used herein, the terms "pharmaceutically acceptable", "1physiologically tolerable" and grammatical variations thereof, *as they refer to co mpositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production .of undesirable physiological effects such as nausea, dizziness, gastric upset and the like..

The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art. Typically such compositions are prepared as sterile injectables either as liquid solutions or suspensions, aqueous or-non-aqueous, however, solid forms suitable-for solution, or suspensions, in liquid prior to use can also be prepared.

The preparation can also be emulsified. *The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, -water, saline, dextrose, glycerol, ethanol or the like and combinations thereof.

in addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the comp onents therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free 32 amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplary of liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, propylene glycol, polyethylene glycol and other solutes.

Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, organic esters such as ethyl.. oleate, and water-oil emulsions. A therapeutic composition contains an HIVneutralizing of a human monoclonal antibody of the present i.

invention, typically an amount of at least 0.1 weight percent of antibody per weight of total therapeutic composition. A weight percent is a ratio by weight of antibody to total composition. Thus, for example, 0.1 weight percent is 0.1 grams of antibody per 100 grams of total composition.

2. Therapeutic Methods In view of the demonstrated HIV neutralizing ability of the human monoclonal antibodies of the present invention, the present disclosure.provides for a method for neutralizing HIV in vitro or in vivo. The 33 method comprises contacting a sample believed to contain HIV with a composition comprising a therapeutically effective amount of a human monoclonal antibody of this invention.

For in vivo modalities, the method comprises administering to the patient a therapeutically effective amolint of a physiologically tolerable composition containing a human monoclonal antibody of the invention.

Thus, in a seventh aspect the invention provides a method for providing passive immunotherapy to HIV disease in a human comprising administering to the human an *immunotherapeutically effective amount of the monoclonal antibody of this invention.

A representative patient for practicing the present passive immunotherapeutic methods is any human exhibiting symptoms of HIV-induced disease, including AIDS or related conditions believed to be caused by HIV infection, and humans at risk of HIV infection. Patients -at risk of infection by HIV include babies of HIV-infected pregnant mothers, recipients of transfusions known to contain HIV, users of HIV contaminated needles, individuals who have participated in high risk sexual activities with known HIV-infected individuals, and the "like risk situations.

In one embodiment, the passive immunization method comprises administering a composition comprising more than one species of a whole immunoglobulin molecule of the first aspect of the invention preferably directed to non- •competing epitopes or directed to distinct serotypes or strains of HIV, as to afford increased effectiveness of the passive immunotherapy.

A therapeutically (immunotherapeutically) effective amount of a human monoclonal antibody is a predetermined amount calculated to achieve the desired effect, to neutralize the HIV present in the sample or in the patient, and thereby decrease the amount of detectable HIV in the sample or patient. In the case of L in vivo- therapies, an effective amount can be measured by 34 0 improvements in one or more symptoms associated with HIVinduced disease occurring in the patient, or by serological decreases in HIV antigens.

Thus, the dosage ranges for the administration of the monoclonal antibodies of the invention are..those large enough to produce the desired effect in which the symptoms of the HIV disease are ameliorated or the likelihood of infection decreased. The dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.

The dosage can be adjusted by the individual physician in the event of any complication.

A therapeutically effective amount of an antibody of this invention is typically an amount of antibody such that when administered in a physiologically tolerable composition is sufficient to achieve a plasma concentration of from about .0.1 microgram.(ug) per milliliter (ml) to about 100 ug/ml, preferably from about 1 ug/ml to about 5 ug/ml, and usually about 5 ug/ml.

Stated differently, the dosage can vary from about 0.1 mg/kg to about 300 mg/kg, preferably from about 0.2 mg/kg ,to about 200 mg/kg, most preferably from about 0.5 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

The human monoclonal antibodies of the invention can be administered parenterally by injection or by gradual infusion over time. Although the HIV infection is typically systemic and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery means are contemplated where there is a likelihood that the tissue targeted contains infectious HIV. Thus, human monoclonal antibodies of the invention can be administered intravenously, intraperitoneally, intramuscularly, -subcutaneously, intracavity, transdermally, and can be delivered by ~,:Szg 35 peristaltic means.

The therapeutic compositions containing a human monoclonal antibody of this invention are conventionally administered intravenously, as by injection of a unit dose, for*example. The term "unit dose". when used in reference to -a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; carrier, or vehicle.

The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be .:AS administered depends on the subject to be treated, capacity of the subject's system to utilize the active **~*,ingredient, and degree of therapeutic effect desired.

Precise amounts of active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each individual. However, suitable dosage ranges for systemic application are disclosed *herein and depend on the route of..administration.

Suitable regimes for administration are al so variable, but -are typified by an initial administration followed by repeated doses at- one or more hour intervals by a subsequent injection or other administration.

Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

As an aid to the administration of effective amounts of a monoclonal antibody, a diagnostic method for detecting a monoclonal antibody in the subject's blood is useful to characterize the fate of the administered therapeutic composition.

The invention also relates to a method for preparing a medicament or pharmaceutical composition comprising the human monoclonal antibodies of the invention, the medicament being used for immunotherapy of 36 HIV disease.

D. Diagnostic Assay Methods The present invention contemplates various assay methods for determining the presence, and preferably amount, of HIV in a sample such as a biological fluid or tissue sample using a human monoclonal antibody of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of HIV in the sample.

In a related embodiment, the present invention contemplates various assay methods for determining the presence, and preferably amount, of an anti-HIV antibody present in a sample such as a biological fluid or tissue sample from -a HIV-infected individual using a human monoclonal antibody of this invention as an immunochemical reagent to form an immunoreaction product whose amount relates, either directly or indirectly, to the amount of .2..20 anti-HIV antibody in the sample.

Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures lin which an immunochemical reagent of

:I

this invention can be used to form an immunoreaction product whose amount relates to the amount of HIV or anti- i HIV antibody present in a body sample. Thus, while exemplary assay methods are described herein, the invention is not so limited.

Various heterogenous and homogeneous protocols, either competitive or noncompetitive, can be employed in performing an assay method of this invention. Examples of types of immunoassays which can utilize monoclonal antibodies of the invention are competitive and noncompetitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay.

Detection of the antigens using the monoclonal antibodies of the invention can be done utilizing immun- L.11. 37 oassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or .can readily discern, other immunoassay formats without undue experimentation.

The monoclonal antibodies of the invention can be bound to many different carriers and used to detect the presence of HIV. Examples, of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, *polyacrylamides, agaroses and magnetite. The nature o f the carrier can be either soluble or insoluble for .purposes of the invention. Those skilled in the art will know of other suitable carriers for binding monoclonal antibodies, or will be able to ascertain such, using routine experimentation.

There are many different labels and methods of labeling known to those of ordinary skill in the art.

Examples-of the types of labels which can be used in the 20 present invention include enzymes, radioisotopes, fluorescent compounds, colloidal-metals, chemiluminescent comounsand bio-luminescent compounds. Those of ordinary skill in the art will know of other suitable labels-for binding to the monocl~onal antibodies of the 25 invention, or will be able to ascertain such, using routine experimentation.. Furthermore, the binding of these labels to the monoclonal antibodies of the 'invention can be done using standard techniques common to those of.

ordinary skill in the art.

For purposes of the invention, HIV may be detected by the monoclonal antibodies of the invention when present in samples of biological fluids and tissues. Any sample containing a detectable amount of HIV can be used. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum and the like, or a solid or semi-solid such as tissues, feces, and the like, or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

38 Another labeling technique which may result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens. These haptens can then be specifically detected by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.

The monoclonal antibodies of the invention are suited for use in vitro, for example, in immunoassays in which theycan be utilized in liquid phase or bound to a solid phase carrier for the detection of HIV in samples, as described above. The monoclonal antibodies in these immunoassays can be detectably labeled in various ways for in vitro use.

In using the human monoclonal antibodies of the invention for the in vivo detection of antigen, the detectably labeled human monoclonal antibody is given in a dose-which is diagnostically effective. The term 20 "diagnostically effective" means that the amount of detectably.,labeled human monoclonal antibody is administered in sufficient quantity to enable detection of the site having the HIV antigen for which the monoclonal antibodies are specific.

The concentration of detectably labeled human monoclonal antibody which is administered should be sufficient such that the binding to HIV is detectable compared to the background. Further, it is desirable that the detectably labeled monoclonal antibody be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labeled human monoclonal antibody for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of human monoclonal antibody can vary from about 0.01 mg/m 2 to about 500 mg/m 2 preferably 0.1 mg/m 2 to about 200 mg/m 2 most preferably about 0.i mg/m 2 to about 10 mg/m 2 Such dosages may vary, SUE 'S&OSSt-&N~- 39 for example, depending on whether multiple injections are given, tissue, and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrument available is a major factor in r selecting a given radioisotope. The radioisotope chosen must have a type of decay which is detectable for a given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized. Ideally, a radioisotope used for in vivo imaging will lack a particle emission, but produce a.large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.

For in vivo diagnosis radioisotopes may be bound to immunoglobulin either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to immunoglobulins are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic 25 acid (EDTA) and similar molecules. Typical examples of metallic, ions which can be bound to the monoclonal antibodies of the invention are 1 'In, 9 Ru, 7 Ga, "Ga, 7As 89 Zr, and 2 0 Tl.

The monoclonal antibodies of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include 57 Gd, 55 Mn, 'Dy, "Cr, and 40 The human monoclonal antibodies of the invention can be used in vitro and in vivo to monitor the course of HIV disease therapy. Thus, for example, by measuring the increase or decrease in the number of cells infected with HIV-or changes in the concentration of HIV present in the body or in various body fluids, it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating the HIV disease is effective.

In a related diagnostic embodiment, the invention contemplates screening HIV-infected patients for the presence of circulating anti-HIV antibodies immunoreactive with gpl20 that have a similar epitope immunospecificity when compared to a neutralizing antibody of this invention. Such a screening method indicates that the HIV-infected patient is exhibiting a significant immune response to the virus, and provides useful information regarding disease status and prognosis. The presence of anti-HIV antibodies cross-reactive with a neutralizing antibody of this invention indicates that the patient has some degree of HIV neutralizing activity, as defined herein.

The diagnostic assay involves determining whether the patient contains human anti-HIV antibodies immunoreactive with the same, similar or overlapping epitopes as a neutralizing antibody of the invent-ionL such :that there is a likelihood that there is a useful neutralizing immune response in the patient. There are a variety of immunological assay formats that can be S* utilized to determine cross-reactivity of test and control antibodies, and the invention need not be so limiting.

Particularly preferred are competition assays for a common antigen, preferably in the solid phase.

A preferred embodiment of the competition immunoassay method comprises the steps of: contacting a sample believed to contain a human anti-HIV antibody with a diagnostically effective JR' amount of the whole immunoglobulin molecule described -fl fi-- 41 herein that binds mature gpl20 in a competition immunoreaction admixture containing mature gpl20 in the solid phase; maintaining said competition immunoreaction admixture under conditions sufficient for said whole immunoglobulin molecule to bind with said gpl20 in the solid phase and form a solid phase immunoreactant; and detecting the amount of said immunoreactant present in said solid phase, and thereby the immunocompetence of any human anti-HIV antibody in said sample.

A diagnostically effective amount, in this context, is a amount relative to the solid phase preferably "mature" gpl20 as defined herein, sufficient to produce a detectable solid phase immunoreaction product between the solid phase gpl20 and the control antibody of this invention. Exemplary competition assays are described herein using the b12 antibody.

Conditions for conducting the competition immunoreaction are well known in the art and can be varied according to recognized parameters in the contacting, the reaction admixtures, the maintenance step, the immunoreaction conditions and the detecting step. For example, the detection step can be conducted by use of a labeled antibody of this invention, by use of a second, labeled anti-human antibody, and the like, as described herein.

E. Diacnostic Systems The present invention also describes a diagnostic system, preferably in kit form, for assaying for the presence of HIV or an anti-HIV antibody in a sample according to the diagnostic methods described herein. A diagnostic system includes, in an amount sufficient to perform at least one assay, a subject human monoclonal antibody, as a separately packaged reagent.

In another embodiment, a diagnostic system is contemplated for assaying for the presence of an anti-HIV Smonoclonal antibody in a body fluid sample such as for 42 -PCTIUS9/08743 monitoring the fate of therapeutically administered antibody. The system includes, in an amount sufficient for at least one assay, a subject antibody as a control reagent, and preferably a preselected amount of HIV ant-igen, each as separately packaged immunochemical reagents.

Instructions for use of the packaged reagent are also typically included.

"Instructions for use" typically include a S. 0 tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/ sample admixtures, temperature, buffer conditions and the like.- In embodiments for detecting HIV or anti-HIV antibody mn-a body fluid, a diagnostic system of the present invention can include a label or indicating means capable of signaling the formation of an immunocomplex containing a human monoclonal antibody of the present 0 :invention.

The word "complex" as used herein *refers to the product of a specific binding reaction such as an antibody-antigen reaction. Exemplary complexe's are immunoreaction products.

As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and mol ecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of-an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.

~Tv$~vv~vm 43 The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), 5-dimethylamine-l-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., eds., John Wiley Sons, Ltd., pp.

189-231 (1982), which is incorporated herein by reference.

15 In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like. In such cases where the principal indicating group is an enzyme such as HRP or glucose oxidase, additional reagents are required to visualize the 20 fact that a receptor-ligand complex (immunoreactant) has formed.. Such additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2' -amino-di- (3-ethyl-benzthiazoline- 25 G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124I, 1251, 1281, 132I and s 5 Cr represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly preferred is 125I.

Another group of useful labeling means are those elements such as 1O and 13N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such 11' indium of 3

H.

Z~A~ >z~V2vt4A~. x2.sllm;4cv z4A~Yr~4g,~~s->- V Vt 44 The linking of labels, labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotopecontaining amino acids provided as a component in the culture medium. See, for example, Galfre et al., Meth.

Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, for example, Aurameas et al., Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a molecular entity capable of selectively binding a reagent species of the present invention or -a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention. Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like. Preferably the specific binding agent binds the reagent species when that species is present as part of a complex.

In preferred embodiments, the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent.

In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent speciescontaining complex.

The diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of an antigen or antibody of this invention in a vascular fluid sample such as blood, serum, or plasma. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzymer 3 'U U+'r 45 antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample.

A

descriptionof the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunologv by D.P. Sites et al., published by Lange Medical Publications of Los Altos, CA in 1982 and in U.S. Patents No.

3,-654,090; No. 3,850,752; and No. 4,016,043, which are -all incorporated herein by reference.

Thus, in some embodiments, a human monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.

A reagent is typically affixed to a solid matrix by adsorption from an aqueous medium although other modes of affixation applicable to proteins and polypeptides well known to those skilled in the art, can be used.

Useful solid matrices are also well known in the i art.. Such materials are water insoluble and include the 20 cross-linked -dextran available under the trademark I SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ); agarose; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott.

Laboratories of North Chicago, IL; polyvinyl chloride, 25 polystyrene, cross-linked polyacrylamide, nitrocelluloseor nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrene or polyvinylchloride.

The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, in lyophilized form. Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.

fe 46 The packaging materials discussed herein.in relation to diagnostic systems are those customarily utilized in diagnostic systems.

The term "package" refers to a solid matrix or material such as glass, plastic polyethylene, polypropylene and polycarbonate), paper, foil and the like capable of holding within fixed limits a diagnostic reagent.such as a monoclonal antibody of the present invention. Thus, for example, a package can be a bottle, vial, plastic and plastic-foil laminated envelope or the like container used to contain a contemplated diagnostic reagent or it can be a microtiter plate well to which microgram quantities of a contemplated diagnostic reagent have been operatively affixed, linked so as to be capable of being immunologically bound by an antibody or polypeptide to be detected.

The materials for use in the assay of the invention are ideally suited for the preparation of a kit.

Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, .each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a human monoclonal antibody of the invention which is, or can be, detectably labelled. The kit may also have containers containing any of the other above-recited immunochemical reagents used to practice the diagnostic methods.

F. Methods for Producing an HIV-Neutralizinq Human Monoclonal Antibody The present invention describes methods for producing novel HIV-neutralizing human monoclonal antibodies. The methods are based generally on the use of combinatorial libraries of antibody molecules which can be produced from a variety of sources, and include naive libraries, modified libraries, and libraries produced directly from human donors exhibiting an HIV-specific 47 immune response.

The combinatorial library production and manipulation methods have been extensively described in the literature, and will not be reviewed in detail herein, except for those feature required to make .and use unique embodiments of the present invention. However, the methods generally involve the use of a filamentous phage (phagemid) surface expression vector system for cloning and expressing antibody species of the library. Various phagemid cloning systems to produce combinatorial libraries have been described by others. See, for example the preparation of combinatorial antibody libraries on phagemids as described by Kang et al., Proc. Natl. Acad.

Sci., USA, 88:4363-4366 (1991); Barbas et al., Proc. Natl.

Acad. Sci., USA, 88:7978-7982 (1991); Zebedee et al., Proc. Natl. Acad. Sci., USA, 89:3175-3179 (1992); Kang et al., Proc. Natl. Acad. Sci USA, 88:11120-11123 (1991); Barbas et al., Proc. Natl. Acad. Sci., USA, 89:4457-4461 i (1992); and Gram et Proc. Natl. Acad. Sci., USA, 20 89:3576-3580 (1992), which references are hereby incorporated by reference.

In one embodiment, the method involves preparing a phagemid library of human monoclonal antibodies by using donor immune cell messenger RNA from HIV-infected donors.

25 The donors can be symptomatic of AIDS, but in preferred embodiments the donor is asymptomatic, as the resulting library contains a substantially higher number of HIVneutralizing human monoclonal antibodies.

In another embodiment, the donor is naive relative to an immune response to HIV, the donor is not HIVinfected. Alternatively, the library can be synthetic, or can be derived from a donor who has an immune response to other antigens.

The method for producing a human monoclonal antibody generally involves preparing separate H and L chain-encoding gene libraries in cloning vectors using human immunoglobulin genes as a source for the libraries, combining the H and L chain encoding gene libraries 48 into a single dicistronic expression vector capable of expressing and assembling a heterodimeric antibody molecule, expressing the assembled heterodimeric antibody molecule on the surface of a filamentous phage particle, isolating the surface-expressed phage particle using immunoaffinity techniques such as panning of phage particles against a preselected antigen, thereby isolating one or more species of phagemid containing i particular H and L chain-encoding genes and antibody *10 molecules that immunoreact with the preselected antigen.

As described herein the Examples, the resulting phagemid library can be manipulated to increase and/or alter the immunospecificities of the monoclonal antibodies of the library to produce and subsequently identify additional, desirable, human monoclonal antibodies of the present invention.

For example, the heavy chain and light (L) chain immunoglobulin molecule encoding genes can be randomly mixed (shuffled) to create new HL pairs in an 20 assembled immunoglobulin molecule. Additionally, either or both the H and L chain encoding genes can be mutagenized in the complementarity determining region (CDR) of the variable region of the immunoglobulin polypeptide, and subsequently screened for desirable immunoreaction and neutralization capabilities.

In one embodiment, the H and L genes can be cloned into separate, monocistronic expression vectors, referred to as a "binary" system described further herein. In this method, step above differs in that the combining of H and L chain encoding genes occurs by the co-introduction of the two binary plasmids into a single host cell for expression and assembly of a phagemid having the surface accessible antibody heterodimer molecule.

In one shuffling embodiment, the shuffling can be accomplished with the binary expression vectors, each capable of expressing a single heavy or light chain encoding gene, as described in Example 11.

4 SZ& 49 In the present methods, the antibody molecules are monoclonal because the cloning methods allow for the preparation of clonally pure species of antibody producing cell lines. In addition, the monoclonal antibodies are human because the H and L chain encoding genes are derived from human immunoglobulin producing immune cells, such as spleen, thymus, bone marrow, and the like.

The method of producing a HIV-neutralizing human monoclonal antibody also requires that the resulting antibody library, immunoreactive with a preselected HIV antigen, is screened for the presence of antibody species which have the capacity to neutralize HIV in one or more of the assays described herein for determining neutralization capacity. Thus, a preferred library of 5 antibody molecules is first produced which binds to an HIV antigen, preferably gpl60, gpl20, gp4l, the V3 loop region of gpl60, or the CD4 binding site of gp120 and gp4l, and then is screened for the-presence of HIV-neutralizing antibodies as described herein.

Additional libraries can be screened from shuffled libraries for additional HIV-immunoreactive and neutralizing human monoclonal antibodies.

As a further characterization of the present invention the nucleotide and corresponding amino acid 25 residue sequence of the antibody molecule's H or L chain encoding gene is determined by nucleic acid sequencing.

The primary amino acid residue sequence information provides essential information regarding the antibody molecule's epitope reactivity.

Sequence comparisons of identified HIVimmunoreactive monoclonal antibody variable chain region sequences are shown herein in Figures 10-13. The Gequences are aligned based on sequence homology, and groups of related antibody molecules are identified thereby in which heavy chain or light chain genes share substantial sequence homology.

An exemplary preparation of a human monoclonal antibody is described in the Examples. The isolation of a S~ VA j;Q4 ~~§4tE SrvzrZ. s~ 50 particular vector capable of expressing an antibody of interest involves the introduction of the dicistronic expression vector into a host cell permissive for expression of filamentous phage genes and the assembly of phage particles. Where the binary vector system is used, both vectors are introduced in the host cell. Typically, the host is E. coli. Thereafter, a helper phage genome is introduced into the host cell containing the immunoglobulin expression vector(s) to provide the genetic complementation necessary to allow phage particles to be assembled. The resulting host cell is cultured to allow the introduced phage genes and immunoglobulin genes to be expressed, and for phage particles to be assembled and shed from the host cell. The shed phage particles are then harvested (collected) from the host cell culture media and screened for desirable immunoreaction and neutralization properties. Typically, the harvested particles are "panned" for immunoreaction with a preselected antigen. The strongly immunoreactive 20 particles are then collected, and individual species of particles are clonally isolated and further screened for SHIV neutralization. Phage which produce neutralizing antibodies are selected and used as a source of a human "HIV neutralizing monoclonal antibody of this invention.

Human monoclonal antibodies of this invention can also be produced by altering the nucleotide sequence of a polynucleotide sequence that encodes a heavy or light chain of a monoclonal antibody of this invention. For example, by site directed mutagenesis, one can alter the nucleotide sequence of an expression vector and thereby introduce changes in the resulting expressed amino acid residue sequence. Thus one can take the polynucleotide of SEQ ID NO 66, for example, and convert it into the polynucleotide of SEQ ID NO 67. Similarly, one can take a known polynucleotide and randomly alter it by random mutagenesis, reintroduce the altered polynucleotide into an expression system and subsequently screen the product H:L pair for HIV-neutralizing activity.

51 Site-directed and random mutagenesis methods are well known in the polynucleotide arts, and are not to be construed as limiting as methods for altering the nucleotide sequence of a subject polynucleotide. Due to the presence of the phage particle in an immunoaffinity isolated antibody, one embodiment involves the-manipulation of the resulting cloned genes to truncate the immunoglobulin-coding gene such that a soluble Fab fragment is secreted by the host E. coli cell containing the phagemid vector. Thus, the resulting manipulated cloned immunoglobulin genes produce a soluble Fab which can be readily characterized in ELISA assays for epitope binding studies, in competition assays with known anti-HIV antibody molecules, and in HIV neutralization assays. The solubilized Fab provides a reproducible and comparable antibody preparation for comparative and characterization studies.

The preparation of soluble Fab is generally described in the immunological arts, and can be conducted as described herein in Example 2b6), or as described by Burton etal., Proc. Natl Acad. Sci., USA, 88:10134-10137 et al (1991) G. Expression Vectors and Polynucleotides for Expressing Anti-HIV Monoclonal Antibodies The preparation of human monoclonal antibodies of this invention depends, in one embodiment, on the cloning and expression vectors used to prepare the combinatorial antibody libraries described herein. The.: cloned immunoglobulin heavy and light chain genes can be shuttled between lambda vectors, phagemid vectors and plasmid vectors at various stages of the methods described herein.

The phagemid vectors produce fusion proteins that are expressed on the surface of an assembled filamentous phage particle.

A preferred phagemid vector of the present invention is a recombinant DNA (rDNA) molecule containing 52 a nucleotide sequence that codes for and is capable of expressing a fusion polypeptide containing, in the direction of amino- to carboxy-terminus, a prokaryotic secretion signal domain, a heterologous polypeptide defining an immunoglobulin heavy or light chain variable region, and a filamentous phage membrane anchor domain. The vector includes DNA expression control sequences for expressing the fusion polypeptide, preferably prokaryotic control sequences.

*.1'10 The filamentous phage membrane anchor is preferably a domain of the cpIII or cpVIII coat protein capable of associating with the matrix of a filamentous phage particle, thereby incorporating the fusion i" polypeptide onto the phage surface.

The secretion signal is a leader peptide domain of a protein that targets the protein to the periplasmic membrane of gram negative bacteria. A preferred secretion signal is a pelB secretion signal. The predicted amino acid residue sequences of the secretion signal domain from 20 two pelB gene product variants from Erwinia carotova are described in Lei et al., Nature, 331:543-546 (1988).

The leader sequence of the pelB protein has previously been used as a secretion signal for fusion proteins (Better et al., Science, 240:1041-1043 (1988); Sastry et al., Proc. Natl. Acad. Sci., USA, 86:5728-5732 (1989); and Mullinax et al., Proc. Natl. Acad. Sci., USA, 87:8095-8099 (1990)). Amino acid residue sequences for other secretion signal polypeptide domains from E. coli useful in this invention as described in Oliver, Escherichia coli and Salmonella Tvphimurium, Neidhard, F.C. American Society for Microbiology, Washington, 1:56.-69 (1987).

*Preferred membrane anchors for the vector are obtainable from filamentous phage M13, fl, fd, and equivalent filamentous phage. Preferred membrane anchor domains are found in the coat proteins encoded by gene III and gene VIII. The membrane anchor domain of a filamentous phage coat protein is a portion of the carboxy X c- i4 53 terminal region of the coat protein and includes a region of hydrophobic amino acid residues for spanning a lipid bilayer membrane, and a region of charged amino acid residues normally found at the cytoplasmic face of the membrane and extending away from the membrane.

In the phage fl, gene VIII coat protein's membrane spanning region comprises residue Trp-26 through and the cytoplasmic region comprises the carboxy-terminal 11 residues from 41 to 52 (Ohkawa et al., J. Biol. Chem., 256:9951-9958 (1981)). An exemplary membrane anchor would consist of residues 26 to 40 of cpVIII. Thus, the.amino acid residue sequence of a preferred membrane anchor domain is derived from the M13 filamentous phage gene VIII coat protein (also designated cpVIII or CP Gene VIII coat protein is present on a mature filamentous phage over the majority of the phage particle with typically about:' 2500 to 3000 copies of the coat protein.

In addition, the amino acid residue sequence of another preferred membrane anchor domain is derived from 20 the M13 filamentous phage gene III coat protein (also Sdesignated cpIII) Gene III coat protein is present on a i: mature filamentous phage at one end of the phage particle with typically about 4 to 6 copies of the coat protein.

For detailed descriptions of the structure filamentous phage particles, their coat proteins and particle assembly, see the reviews by Rached et al., Microbiol. Rev., 50:401-427 (1986); and Model et al., in "The Bacteriophages: Vol. R. Calendar, ed. Plenum Publishing Co., pp. 375-456 (1988).

DNA expression control sequences comprise a set of DNA expression signals for expressing a structural gene product and include both 5' and 3' elements, as is well known, operatively linked to the cistron such that the cistron is able to express a structural gene product. The 5' control sequences define a promoter for initiating transcription and a ribosome binding site operatively linked at the 5' terminus of the upstream translatable DNA sequence.

S~ 54 To achieve high levels of gene expression in E.

coli, it is necessary to use not only strong promoters to generate large quantities of mRNA, but also ribosome binding sites to ensure that the mRNA is efficiently translated. In E. coli, the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-11 nucleotides upstream from the initiation codon (Shine et al., Nature, 254:34 (1975).

The sequence, AGGAGGU, which is called the Shine-Dalgarno (SD) sequence, is complementary to the 3' end of E. coli 16S rRNA. Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors: The degree of complementarity between the SD sequence and 3' end of the 16S rRNA.

(ii) The spacing and possibly the DNA sequence lying between the SD sequence and the AUG.

Roberts et al., Proc. Natl. Acad. Sci.. USA, 76:760, (1979a); Roberts et al., Proc. Natil. Acad. Sci. USA, 76:5596 (1979b); Guarente et al., Science, 209:1428 (1980); and Guarente et al., Cell, 20:543 (1980).

Optimization is achieved by measuring the level of expression of genes in plasmids. in which this spacing is S systematically altered. Comparison of different mRNAs shows that there are statistically preferred sequences from positions -20 to +13 (where the A of the AUG is position Gold et al., Annu. Rev. Microbl., 35:365 (1981). Leader sequences have been shown to influence translation dramatically. Roberts et al., 1979 a, b supra.

(iii) The nucleotide sequence following the AUG. which affects ribosome binding. Taniguchi et al., J Mol. Biol., 118:533 (1978).

The 3' control sequences define at least one termination (stop) codon in frame with and operatively linked to the heterologous fusion polypeptide In preferred embodiments, the vector utilized includes a prokaryotic origin of replication or replicon, a DNA sequence having the ability to direct 55 autonomous replication and maintenance of the recombinant DNA molecule extra chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such origins of replication are well known in the art. Preferred origins of replication are those that are efficient in the host organism. A preferred host cell is-E. coli. For use of a vector in E. coli, a preferred origin of replication is ColE1 found in pBR322 and a variety of other common plasmids. Also preferred is the pl5A origin of replication found on pACYC and its derivatives. The ColE1 and pl5A replicon have been :extensively utilized in molecular biology, are available on a variety of plasmids and are described at least by 'Sambrook et al., in "Molecular Cloning: a Laboratoryi Manual", 2nd edition, Cold Spring Harbor Laboratory Press (1989) The ColE1 and pl5A replicons are particularly preferred for use in one embodiment of the present invention where two "binary" plasmids are utilized because they each have the ability to direct the replication of plasmid in E. coli while the other.replicon is present in a second plasmid in the same E. coli cell. In other words, ColE1 and pl5A are non-interfering replicons that allow the maintenance of two plasmids in the same host ii (see, for example, Sambrook et al., supra, at pages 1.3- This feature is particularly important in the binary vectors embodiment of the present invention because a single host cell permissive for phage replication must support the....independent and simultaneous replication of two separate vectors, namely a first vector for expressing a heavy chain polypeptide, and a second vector- for expressing a light chain polypeptide.

In addition, those embodiments that include a prokaryotic replicon can also include a gene whose expression confers a selective advantage, such as drug resistance, to a bacterial host transformed therewith. Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, -i r 56 neomycin/kanamycin or cholamphenicol. Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences. Exemplary vectors are the plasmids pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories, (Richmond, CA) and pPL and pKK223 available from Pharmacia, (Piscataway, NJ) A vector for expression of a monoclonal antibody of the invention on the surface of a filamentous phage particle is a recombinant DNA (rDNA) molecule adapted for 10 receiving and expressing translatable first and second DNA sequences in the form of first and second polypeptides wherein one of the polypeptides is fused to a filamentous

S.-

phage coat protein membrane anchor. That is, at least one of the polypeptides is a fusion polypeptide containing a filamentous phage membrane anchor domain, a prokaryotic secretion signal domain, and an immunoglobulin heavy or light chain variable domain.

A DNA expression vector for expressing a heterodimeric antibody molecule provides a system for independently cloning (inserting) the two translatable DNA sequences into two separate cassettes present in the vector, to form two separate cistrons for expressing the ;first and second polypeptides of the antibody molecule, or the ligand binding portions of the polypeptides that comprise the antibody molecule the H and L variable regions of an immunoglobulin molecule). The DNA I" expression vector for expressing two cistrons is referred to as a dicistronic expression vector.

The vector comprises a first cassette that includes upstream and downstream translatable DNA sequences operatively linked via a sequence of nucleotides adapted for directional ligation to an insert DNA. The upstream translatablesequence encodes the secretion signal as defined herein. The downstream translatable sequence encodes the filamentous phage membrane anchor as defined herein. The cassette preferably includes DNA expression control sequences for expressing the receptor polypeptide that is produced when an insert translatable .z~Q 57 DNA sequence (insert DNA) is directionally inserted into the cassette via the sequence of nucleotides adapted for directional ligation. The filamentous phage membrane anchor is preferably a domain of the cpIII or cpVIII coat protein capable of binding the matrix of a filamentous phage particle, thereby incorporating the fusion polypeptide onto the phage surface.

The receptor expressing vector also contains a second cassette for expressing a second receptor polypeptide. The second cassette includes a second translatable DNA sequence that encodes a secretion signal, as defined herein, operatively linked at its 3' terminus via a sequence of nucleotides adapted for directional ligation to a downstream DNA sequence of the vector that typically defines at least one stop codon in the reading frame of the cassette. The second translatable DNA sequence is operatively linked at its 5' terminus to DNA expression control sequences forming the 5' elements. The second cassette is capable, upon insertion of a translatable DNA sequence (insert DNA), of expressing the second fusion polypeptide comprising a receptor of the secretion signal with a polypeptide coded by the insert DNA. .l" An upstream translatable DNA sequence encodes a :25 prokaryotic secretion signal as described earlier. The upstream translatable DNA sequence encoding the pelB secretion signal is a preferred DNA sequence for inclusion in a receptor expression vector. A downstream translatable DNA sequence encodes a filamentous phage membrane anchor as described earlier. Thus, a downstream translatable DNA sequence encodes an amino acid residue i sequence that corresponds, and preferably is identical, to the membrane anchor domain of either a filamentous phage gene III or gene VIII coat polypeptide.

A cassette in a DNA expression vector of this invention is the region of the vector that forms, upon insertion of a translatable DNA sequence (insert DNA), a sequence of nucleotides capable of expressing, in an 58 appropriate host, a fusion polypeptide. The expressioncompetent sequence of nucleotides is referred to as a cistron. Thus, the cassette comprises DNA expression control elements operatively linked to the upstream and downstream translatable DNA sequences. A cistron is formed when a translatable DNA sequence is directionally inserted (directionally ligated) between the upstream and downstream sequences via the sequence of nucleotides adapted for that purpose. The resulting three 10. translatable DNA sequences, namely the upstream, the inserted and the downstream sequences, are all operatively linked in the same reading frame.

Thus,. a DNA expression vector for expressing an antibody molecule provides a system for cloning translatable DNA sequences into the cassette portions of the vector to produce cistrons capable of expressing the first and second polypeptides, the heavy and light chains of a monoclonal antibody.

As used herein, the term "vector" refers to a nucleic acid molecule capable of. transporting between different genetic environments another nucleic acid to which it has been operatively linked. Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operatively linked. Vectors, therefore, preferably contain the replicons and selectable markers described earlier.

As used herein with regard to DNA sequences or segments, the phrase "operatively linked" means the sequences or segments have-been covalently joined, preferably by conventional phosphodiester bonds, into one strand of DNA, whether in single or double stranded form.

The choice of vector to which transcription unit or a cassette of this invention is operatively linked depends directly, as is well known in the art, on the functional properties desired, vector replication and protein expression, and the host cell to be transformed, these being limitations inherent in the art of constructing t 59 recombinant DNA molecules.

A sequence of nucleotides adapted for directional ligation, a polylinker, is a region of the DNA expression vector that operatively links for replication and transport the upstream and downstream il translatable DNA sequences and provides a site or means for directional ligation of a DNA sequence into the vector. Typically, a directional polylinker is a sequence of nucleotides that defines two or more restriction endonuclease recognition sequences, or restriction sites.

Upon restriction cleavage, the two sites yield cohesive termini to which a translatable DNA sequence can be ligated to the DNA expression vector. Preferably, the two restriction sites provide, upon restriction cleavage, '15 cohesive termini that are non-complementary and thereby .i,!ip| permit directional insertion of a translatable

DNA

sequence into the cassette. In one embodiment, the directional ligation means is provided by nucleotides present, in the upstream translatable DNA sequence, downstream translatable DNA sequence, or both. In another embodiment, the sequence of nucleotides adapted for directional ligation comprises a sequence of nucleotides that defines multiple directional cloning means. Where the sequence of nucleotides adapted for directional ligation'defines numerous restriction sites, it is referred to as a multiple cloning site.

In a preferred embodiment, a DNA expression vector is designed for convenient manipulation in the form of a filamentous phage particle encapsulating a genome according to the teachings of the present invention. In this embodiment, a DNA expression vector further contains a nucleotide sequence .that defines a filamentous phage origin of replication such that the vector, upon presentation of the appropriate genetic complementation, can replicate as a filamentous phage in single stranded replicative form and be packaged into filamentous phage particles. This feature provides the ability of the DNA expression vector to be packaged into phage particles for Jar- 60 subsequent segregation of the particle, and vector contained therein, away from other particles that comprise a population of phage particles.

A filamentous phage origin of replication is a region of the phage genome, as is well known, that defines sites for initiation of replication, termination of replication and packaging of the replicative form produced by replication (see, for example, Rasched et al., Microbiol. Rev., 50:401-427 (1986); and Horiuchi, J. Mol. Biol., 188:215-223 (1986)).

A preferred filamentous phage origin of replication for use in the present invention is an M13, fl or fd phage origin of replication (Short et al., Nucl.

Acids Res., 16:7583-7600 (1988)). Preferred DNA 5 expression vectors for cloning and expression a human monoclonal antibody of this invention are the dicistronic expression vectors pComb8, pComb2-8, pComb3, pComb2-3 and pComb2-3', described herein.

A particularly preferred vector of the present invention includes a polynucleotide sequence that encodes a heavy or light chain variable region of a human '.t monoclonal antibody of the present invention.

Particularly preferred are vectors that include a nucleotide sequence that encodes a heavy or light chain amino acid residue sequence shown in Figures 10-13, that encodes a heavy or light chain having the binding specificity of those sequences shown in Figures 10-13, or that encodes a heavy or light chain having conservative substitutions relative to a sequence shown in Figures 13, and complementary polynucleotide sequences thereto.

Insofar as polynucleotides are component parts of a DNA expression vector for producing a human monoclonal antibody heavy or light chain immunoglobulin variable region amino acid residue sequence, the invention also contemplates isolated polynucleotides that encode such heavy or light chain sequences.

It is to be understood that, due to.the genetic code and its attendant redundancies, numerous I iX- 61 polynucleotide sequences can be designed that encode a contemplated heavy or light chain immunoglobulin variable region amino acid residue sequence. Thus, the invention contemplates such alternate polynucleotide sequences incorporating the features of the redundancy of the genetic code.

Insofar as the expression vector for producing a human monoclonal antibody of this invention is carried in a host cell compatible with expression of the antibody, the invention contemplates a host cell containing a vector or polynucleotide of this invention. A preferred host cell is E. coli, as described herein.

E. coli cultures containing preferred expression vectors that produce a human monoclonal antibody of this invention were deposited pursuant to Budapest Treaty requirements with the American Type Culture Collection (ATCC),.Rockville, MD, as described herein.

.ii; Examples The following examples are intended to illustrate, but not limit, the scope of the invention. 1. Construction of a Dicistronic Expression Vector for Producing a Heterodimeric Receptor on Phage i :25 Particles To obtain a vector system for generating a large number of Fab antibody fragments that can be screened.

directly, expression libraries in bacteriophage Lambda have previously been constructed as described in Huse et al., Science, 246:1275-1281 (1989). These systems did not contain design features that provide for the expressed Fab to be targeted to the surface of a filamentous phage particle.

The main criterion used in choosing a vector 35 system was the necessity of generating the largest number of Fab fragments which could be screened directly.

Bacteriophage Lambda was selected as the starting point to develop an expression vector for three reasons. First, in 1. .ll:r rnl~ i~l;r ;i 62 vitro packaging of phage DNA was the most efficient method of reintroducing DNA into host cells. Second, it was possible to detect protein expression at the level of single phage plaques. Finally, the screening of phage libraries typically involved less difficulty with nonspecific binding. The alternative, plasmid cloning vectors, are only advantageous in the analysis of clones after they have been identified. This advantage was not lost in the present system because of the use of a 10 dicistronic expression vector such as pCombVIII, thereby permitting a plasmid containing the heavy chain, light chain, or Fab expressing inserts to be excised.

.*o a. Construction of Dicistronic Expression Vector pCOMB 0 1) Preparation of Lambda ZaO TMII Lambda ZapTM II is a derivative of the "original Lambda Zap (ATCC Accession No. 40,298) that maintains all of the characteristics of the original Lambda Zap including 6 unique cloning sites, fusion protein expression, and the ability to rapidly excise the insert in the form of a phagemid (Bluescript but lacks the SAM 100 mutation, allowing growth on many Non-Sup F strains, including XLl-Blue. The Lambda ZapTM II was constructed as described in Short et al., Nuc. Acids Res., 16:7583-7600, 1988, by replacing the Lambda S gene contained in a 4254 base pair (bp) DNA fragment produced by digesting Lambda Zap with the restriction enzyme Nco I.

This 4254 bp DNA fragment was replaced with the 4254 bp DNA fragment containing the Lambda S gene isolated from Lambda gtlO (ATCC 40,179) after digesting the vector with the restriction enzyme Nco I. The 4254 bp DNA fragment isolated from lambda gtlO was ligated into the original Lambda Zap vector using T4 DNA ligase and standard protocols such as those described in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, NY, 1987, to form Lambda ZapTM II.

63 2) Preparation of Lambda Hc2 To express a plurality of VH-coding DNA homologs in an E. coli host cell, a vector designated Lambda Hc2 was constructed. The vector provided the following: the capacity to place the VH-coding

DNA

homologs in the proper reading frame; a ribosome binding site as described by Shine et al., Nature, 254:34 (1975); a leader sequence directing the expressed protein to the periplasmic space designated the pelB secretion signal; a polynucleotide sequence that coded for a known epitope (epitope tag); and also a polynucleotide that coded for a spacer protein between the VH-coding DNA homolog and the polynucleotide coding for the epitope tag. Lambda Hc2 has been previously described by Huse et al., Science, 15 246:1275-12.81 (1989) To prepare Lambda Hc2, a synthetic DNA sequence containing all of the above features was constructed by designing single stranded polynucleotide segments of 20-40 bases that would hybridize to each other and form the double stranded synthetic DNA sequence shown in Figure 1; The individual single-stranded polynucleotide segments are 9 shown in Table 1.

Polynucleotides -N2, N3, N9-4, N11, N10-5, N6, N7 and N8 (Table 1) were kinased by adding 1 1p of each 25 polynucleotide 0.1 micrograms/microliter (pg/pl) and units of T, polynucleotide kinase to a solution containing mM Tris-HCl (Tris[hydroxymethyl] aminomethane hydrochloride) at pH 7.6,.10 mM MgC1,, 5 mM dithiothreitol (DTT), 10 mM beta-mercaptoethanol, 500 micrograms per milliliter (pg/ml) bovine serum albumin (BSA). The solution was maintained at 37 degrees Centigrade (37 0

C)

for 30 minutes and the reaction stopped by maintaining the solution at 65 0 C for 10 minutes. The two end polynucleotides, 20 nanograms (ng) of polynucleotides N1 and polynucleotides N12, were added to the above kinasing reaction solution together with 1/10 volume of a solution, containing 20 mM Tris-HCl at pH 7.4, 2.0 mM MgC 2 1 and 50 mM NaCl. This solution was heated to 70 0 C for 5 minutes and I- 64 allowed to cool to room temperature, approximately 25 0

C,

over 1.5 hours in a 500 ml beaker of water. During this time period all 10 polynucleotides annealed to form the double stranded synthetic DNA insert shown in Figure 1.

The individual polynucleotides were covalently linked to each other to stabilize the synthetic DNA insert by adding gl of the above reaction to a solution containing 50 mM Tris-HCl. at pH 7.5, 7 mM MgCl 2 1 mM DTT, 1 mM adenosine triphosphate (ATP) and 10 units of T4 DNA ligase. This solution was maintained at 37 0 C for 30 minutes and then the T4 DNA ligase was inactivated by maintaining the solution at 65 0 C for 10 minutes. The end polynucleotides were kinased by mixing 52 pl of the above reaction, 4 L1 of a solution containing 10 mM ATP and 5 units of T4 polynucleotide kinase. This solution was maintained at 37 0 C for 30 minutes and then the T4 polynucleotide kinase g* was inactivated by maintaining the solution at 65 0 C for minutes.

oo.. Table 1

SEQ

o ID NO (15) N1) 5' GGCCGCAAATTCTATTTCAAGGAGACAGTCAT 3' (16) N2) 5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3' (17) N3) 5' GTTATTACTCGCTGCCCAACCAGCCATGGCCC 3' (18) N6) 5' CAGTTTCACCTGGGCCATGGCTGGTTGGG 3' (19) N7) 5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3' (20) N8) 5' GTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC 3' (21) N9-4) 5' AGGTGAAACTGCTCGAGATTTCTAGACTAGTTACCCGTAC 3' (22) N10--5) 5' CGGAACGTCGTACGGGTAACTAGTCTAGAAATCTCGAG 3! (23) N11) 5' GACGTTCCGGACTACGGTTCTTAATAGAATTCG 3' (24) N12) 5' TCGACGAATTCTATTAAGAACCGTAGTC 3' -iu: I~ rs:; The completed synthetic DNA insert was ligated directly into the Lambda ZapT M II vector described in Example lal) that had been previously digested with the restriction enzymes, Not I and Xho I. The ligation mixture was packaged according to the manufacture's instructions using Gigapack II Gold packing extract available from Stratagene, La S. S tz~=~s 66 Jolla, California. The packaged ligation mixture was plated on XL1-Blue cells (Stratagene).

Individual lambda plaques were cored and the inserts excised according to the in vivo excision protocol for Lambda ZapT II provided by the manufacturer (Stratagene). This in vivo excision protocol moved the cloned insert from the Lambda Hc2 vector into a phagemid vector to allow easy for manipulation and sequencing. The accuracy of the above cloning steps was confirmed by sequencing the insert using the Sanger dideoxy method described in by Sanger et al., Proc. Natl. Acad. Sci., USA, 74:5463-5467 (1977) and using the manufacture's instructions in the AMV Reverse Transcriptase 35 S-ATP sequencing kit (Stratagene). The sequence of the resulting double-stranded synthetic DNA insert in the V H expression vector (Lambda Hc2) is shown in Figure 1. The sequence of each strand (top and bottom) of Lambda Hc2 is listed in the 2.0 Sequence Listing as SEQ ID NO- 1 and SEQ ID NO 2, respectively. The resultant Lambda Hc2 expression vector is shown in Figure 2.

3) Preparation of Lambda Lc2 To express a plurality of VL-coding DNA homologs in an E. coli host cell, a vector designated Lambda Lc2 was constructed having the capacity to place the VL-coding DNA homologs in the proper reading frame, provided a ribosome binding site as described by Shine et al., Nature, 254:34 (1975), provided the pelB gene leader sequence secretion signal that has been previously used to successfully secrete Fab fragments in E. coli by Lei et al., J. Bac., 169:4379 (1987) and Better et al., Science, 240:1041 (1988), and also provided a polynucleotide containing a restriction endonuclease site for cloning. Lambda Lc2 has been previously described by Huse et al., Science, 4 Y~ SXZ~ThC S3V 67 246:1275-1281 (1989).

A synthetic DNA sequence containing all of the above features was constructed by designing single stranded polynucleotide segments of 20-60 bases!:: that would hybridize to each other and form the double stranded synthetic DNA sequence shown in -Figure 3. The sequence of each individual single-stranded polynucleotide segment (01-08) within the double stranded synthetic DNA sequence is shown in Table 2.

Polynucleotides 02, 03, 04, 05,.06 and 07 (Table 2) were kinased by adding 1 pl (0.1 p g/ l) of each polynucleotide and 20 units of T 4 polynucleotide kinase to a solution containing :"15 mM Tris-HCl at pH 7.6, 10 mM MgC1 2 5 mM DTT, 10 mM beta-mercaptoethanol, 500 Ag/ml of BSA. The solution was maintained at 37°C for 30 minutes and the reaction stopped by maintaining the solution at j 0 C for 10 minutes. The 20 ng each of the two end :.20 polynucleotides, 01 and 08, were added to the above *kinasing reaction solution together with 1/10 ;ii volume of a solution containing 20.0 mM Tris-HC1 at pH 7.4, 2.0 mM MgC1, and 15.0 mM sodium chloride (NaC) This solution was heated to 70 0 C for minutes and allowed to cool to room temperature, approximately 25 0 C, over 1.5 hours in a 500 ml beaker of water. During this time period all 8 polynucleotides annealed to form the double stranded synthetic DNA insert shown in Figure 3.

The individual polynucleotides were covalently linked to each other to stabilize the synthetic DNA insert by adding 40 pl of the above reaction to a solution containing 50 mM Tris-HC1 at pH 7.5, 7 mM MgC1 2 1 mM DTT, 1 mM ATP and 10 units of T4 DNA ligase. This solution was maintained at 37 0 C for minutes and then the T4 DNA ligase was inactivated by maintaining the solution at 65 0 C for minutes. The end polynucleotides were kinased 7 68 by mixing -52 1p of the above reaction, 4 pl of a solution containing 10 mM ATP and 5 units of T4 polynucleotide kinase. This solution was maintained at 37 0 C for 30 minutes and then the T4 polynucleotide kinase was inactivated by maintaining the solution at 65 0 C for 10 minutes.

Table 2

SEQ

ID NO (25) 01) 5' TGAATTCTAAACTAGTCGCCAAGGAGACAGTCAT 3' (26) 02) 5' AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3' (27) 03) 5' GTTATTACTCGCTGCCCAACCAGCCATGGCC 3' (28) 04) 5' GAGCTCGTCAGTTCTAGAGTTAAGCGGCCG 3 (29) 05) 5' GTATTTCATTATGACTGTCTCCTTGGCGACTAGTTTAGAA- TTCAAGCT 3' (30) 06) 5' CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3' (31) 07) TGACGAGCTCGGCCATGGCTGGTTGGG 3' S" (32) 08) 5' TCGACGGCCGCTTAACTCTAGAAC 3' 20 The completed synthetic DNA insert was ligated directly into the Lambda ZapT' II vector described in Example lal) that had been previously digested with the restriction enzymes Sac I and Xho I. The ligation mixture was packaged according to the manufacture's instructions using Gigapack II Gold packing extract (Stratagene). The packaged.

ligation mixture was plated on XL1-Blue cells (Stratagene). Individual lambda plaques were cored and the-inserts excised according to the in vivo excision protocol for Lambda ZapTM II provided by the manufacturer (Stratagene). This in vivo excision protocol moved the cloned insert from the Lambda Lc2 vector into a plasmid phagemid vector allow for easy manipulation and sequencing. The accuracy of the above cloning steps was confirmed A 69 by sequencing the insert using the manufacture's instructions in the AMV Reverse Transcriptase 35 S-dATP sequencing kit (Stratagene). The sequence of the resulting Lc2 expression vector (Lambda Lc2) is shown in Figure 3. Each strand is separately .listed in the Sequence Listing as SEQ ID NO 3 and -SEQ ID NO 4. The resultant Lc2 vector is schematically diagrammed in Figure .4.

A preferred vector for use in this invention, designated Lambda Lc3, is a derivative of Lambda.

Lc2 prepared above. Lambda Lc2 contains a Spe I restriction site located 3' to the EcoR I restriction site and 5' to the Shine-Dalgarno ribosome binding site as shown in the sequence in "15 Figure 3 and in SEQ ID NO 3. A Spe I restriction site is also present in Lambda Hc2 as shown in Figures 1 and 2 and in SEQ ID NO 1. A combinatorial vector, designated pComb, was constructed by combining portions of Lambda Hc2 and Lc2 together as described in Example la4) below.

The resultant combinatorial pComb vector contained two Spe I restriction sites, one provided by Lambda Hc2 and one provided by Lambda Lc2, with an EcoR I site in between. Despite the presence of two Spe I l .restriction sites, DNA homologs having Spe I and EcoR I cohesive termini were successfully directionally ligated into a pComb expression vector previously digested with Spe I and EcoR I as described in Example lb below. The proximity of the EcoR I restriction site to the 3' Spe I site,.

provided by the Lc2 vector, inhibited the complete digestion of the 3' Spe I site. Thus, digesting pComb with Spe I and EcoR I did not result in removal of the EcoR I site between the two Spe I sites.

The presence of a second Spe I -restriction site may be undesirable for ligations into a pComb vector digested only with Spe I as the region r r r i 70 between the two sites would be eliminated.

Therefore, a derivative of Lambda Lc2 lacking the second or 3' Spe I site, designated Lambda Lc3, was produced by first digesting Lambda Lc2 with Spe I to form a linearized vector. The ends were filled in to form blunt ends which are ligated together to result in Lambda Lc3 lacking a Spe I site. Lambda Lc3 is a preferred vector for use in constructing a combinatorial vector as described below.

4) Preparation of pComb Phagemids were excised from the expression vectors Lambda Hc2 or Lambda Lc2 using an in vivo excision protocol described above.

Double stranded DNA was prepared from the phagemid-containing cells according to the methods described by Holmes et al., Anal. Biochem., 114:193 (1981). The phagemids resulting from in vivo excision contained the same nucleotide sequences for antibody fragment cloning and expression as did the parent vectors, and are designated phagemid Hc2 and Lc2, corresponding to Lambda Hc2 and Lc2, :respectively.

For the construction of combinatorial phagemid vector pComb, produced by combining portions of phagemid Hc2 and phagemid Lc2, phagemid Hc2 was first digested with Sac I to remove the restriction site located 5' to the LacZ promoter. The linearized phagemid was then blunt ended with T4 polymerase and ligated to result in a Hc2 phagemid lacking a Sac I site. The modified Hc2 phagemid and the Lc2 phagemid were then separately restriction digested with Sca I and EcoR I to result in a Hc2 fragment having from 5' to 3' Sca I, Not I, Xho I, Spe I and EcoR I restriction sites and a Lc2 fragment having from 5' to 3' EcoR I, Sac I, Xba I and Sac I restriction sites. The linearized phagemids were then ligated together at 71 their respective cohesive ends to form pComb, a circularized phagemid having a linear arrangement of restriction sites of Not I, Xho I, Spe I, EcoR i I,.Sac I, Xba I, Not I, Apa I and Sca I. The ligated phagemid vector was then inserted into an Sappropriate bacterial host and transformants were selected on the antibiotic ampicillin.

Selected ampicillin resistant transformants were screened for the presence of two Not I sites.

The resulting ampicillin resistant combinatorial phagemid vector was designated pComb, the schematic organization of which is shown in Figure 5. The resultant combinatorial vector, pComb, consisted of a DNA molecule having two cassettes to express two :i?5 fusion proteins and having nucleotide residue sequences for the following operatively linked elements listed in a 5' to 3' direction: a first cassette consisting of an inducible LacZ promoter: il upstream from the LacZ gene;.a Not I restriction .":020 site; a ribosome binding site; a pelB leader; a spacer; a cloning region bordered by a 5' Xho and 3' Spe I restriction site; a decapeptide tag followed by expression control stop sequences; an.

EcoR I restriction site located 5' to a second cassette consisting of an expression control ribosome binding site; a pelB leader; a spacer region; a cloning region bordered by a 5' Sac I and a 3' Xba I restriction site followed by expression control stop sequences and a second Not I restriction site.

A preferred combinatorial vector for use in this invention, designated pComb2, is constructed by combining portions of phagemid Hc2 and phagemid Lc3 as described above for preparing pComb. The resultant combinatorial vector, pComb2, consists of a DNA molecule having two cassettes identical to pComb to express two fusion proteins identically to pComb except that a second Spe I restriction site i^~ 72 in the second cassette is eliminated.

b. Construction of the pCombIII Vector for Expressing Fusion Proteins Having a Bacteriophaqe Coat Protein Membrane Anchor Because of the multiple endonuclease restriction cloning sites, the pComb phagemid expression vector prepared above is a useful cloning vehicle for modification for the preparation of an expression vector for use in this o invention. To that end, pComb was digested with EcoR I and Spe I followed by phosphatase treatment to produce linearized pComb.

*0* 1) Preparation of pCombIII A separate phagemid expression vector was constructed using sequences encoding bacteriophage cpIII membrane anchor domain. A PCR S2 D product defining the DNA sequence encoding the filamentous phage coat protein,cpIII, membrane anchor containing a LacZ promotor region sequence 3' to the membrane anchor for expression of.the light chain and Spe I and EcoR I cohesive termini was prepared from M13mpl8, a commercially available bacteriophage vector (Pharmacia, Piscataway, New Jersey).

To prepare a modified cpIII, replicative form DNA .from M13mpl8 was first isolated. Briefly, into 2 ml of LB (Luria-Bertani medium), 50 Al of a culture-of a bacterial strain carrying an F' episome (JM107, JM109 or TG1') was admixed with a one tenth suspension of bacteriophage particles derived from a single plaque. The admixture was incubated for 4 to 5.hours at 37 0 C with constant agitation. The admixture was then centrifuged at 12,000 x g for 5 minutes to pellet the infected bacteria. After the supernatant was removed, the i- 73 pellet was resuspended by vigorous vortexing in 100 pl of ice-cold solution I. Solution I was prepared by admixing 50 mM glucose, 10 mM EDTA (disodium ethylenediaminetetraacetic acid) and 25 mM Tris-HCl at pH 8.0, and autoclaving for 15 minutes.

To the bacterial suspension, 200 1l of freshly -prepared Solution II was admixed and the tube was rapidly inverted five times. Solution II was prepared by admixing 0.2 N NaOH and 1% SDS. To the bacterial suspension, 150 1l of ice-cold Solution III was admixed and the tube was vortexed gently-in an inverted position for 10 seconds to disperse Solution III through the viscous bacterial lysate.

Solution III was prepared by admixing 60 ml of 5 M 15 potassium acetate, 11.5 ml of glacial acetic acid and 28.5 ml of water. The resultant bacterial i lysate was then stored on ice for 5 minutes followed by centrifugation at 12,000 x g for minutes at 4 0 C in a microfuge. The resultant :20 supernatant was recovered and transferred to a new Stube. To the supernatant was added an equal volume of phenol/chloroform and the admixture was vortexed. The admixture was then centrifuged at 12,000 x g for 2 minutes in a microfuge. The .resultant supernatant was transferred to a new tube and the double-stranded bacteriophage DNA was precipitated with 2 volumes of ethanol at room temperature. After allowing the admixture to stand at room-temperature for 2 minutes, the admixture was centrifuged to pellet the DNA. The supernatant was removed and the pelleted replicative form DNA was resuspended in 25 p1 of Tris-HC1 at pH 7.6, and mM EDTA (TE).

An alternative Lac-B primer for use in constructing the cpIII membrane anchor and LacZ promotor region was Lac-B' as shown in Table 3.

The amplification reactions were performed as described above with the exception that in the 74 second PCR amplification, Lac-B' was used with Lac-F instead of Lac-B. The product from the amplification reaction is listed in the sequence listing as SEQ ID NO 41 from nucleotide position 1 to nucleotide position 172. The use of Lac-B' resulted in a LacZ region lacking 29 nucleotides on the 3' end but was functionally equivalent to the longer fragment produced with the Lac-F and Lac-B primers.

The products of the first and second PCR amplifications using the primer pairs G-3 and G-3(B) and Lac-F and Lac-B were then recombined at the nucleotides corresponding to cpIII membrane anchor overlap and Nhe I restriction site and l5 subjected to a second round of PCR using the G-3(F) (SEQ ID NO 35) and Lac-B (SEQ ID NO 38) primer pair to form a recombined PCR DNA fragment product consisting of the following: a 5' Spe I restriction S• site; a cpIII DNA membrane anchor domain beginning at the nucleotide residue sequence which corresponds to the amino acid residue 198 of the entire mature cpIII protein; an endogenous stop site provided by the membrane anchor at amino acid residue number 112; a Nhe I restriction site, a LacZ promoter, operator and Cap-binding site sequence; and a 3' EcoR I restriction site.

To construct a phagemid vector for the coordinate expression of a heavy chain-cpIII fusion protein as prepared in Example 2 with kappa light chain, the recombined PCR modified cpIII membrane anchor domain DNA fragment was then restriction digested with Spe I and EcoR I to produce a DNA fragment for directional.ligation into a similarly digested pComb2 phagemid expression vector having only one Spe I site.prepared in Example la4) to form a pComb2-III (also referred to as pComb2-III) phagemid expression vector. Thus, the resultant ampicillin resistance conferring pComb2-3 vector, 4~-~gkr~.s~4 75 having only one Spe I restriction site, contained separate LacZ promoter/operator sequences for directing the separate expression of the heavy chain (Fd)-cpIII fusion product and the light chain protein. The expressed proteins were directed to _the periplasmic space by pelB leader sequences for -functional assembly on the membrane. Inclusion of the phage Fl intergenic region in the vector allowed for packaging of single stranded phagemid with the aid of helper phage. The use of helper phage superinfection lead to expression of two forms of cpIII. Thus, normal phage morphogenesis was perturbed by competition between the Fab-cpIII fusion and the native cpIII of the helper phage for incorporation into the virion for Fab-cpVIII fusions. In addition, also contemplated for use in ii Sthis invention are vectors conferring chloramphenicol resistance and the like.

A more preferred phagemid expression vector for use in this invention.having additional restriction enzyme cloning sites, designated pComb-III' or-pComb2-3', was prepared as described above for pComb2-3 with the addition of a 51 base pair fragment from pBluescript as described by Short et al., Nuc. Acids Res., 16:7583-7600 (1988) and commercially available from Stratagene. To..

prepare pComb2-3 1 pComb2-3 was first digested with i Xho I and Spe I restriction enzymes to form a linearized pComb2-3; The vector pBluescript was digested with the same enzymes releasing a 51 base pair fragment containing the restriction enzyme sites Sal I, Acc I, Hinc II, Cla I, Hind III, EcoR V, Pst I, Sma I and BamH I. The 51 base pair fragment was ligated into the linearized pComb2-3 vector via the cohesive Xho I and Spe I termini to form pComb2-3'.

76 Table 3

SEQ

ID NO Primer (35)1 G-3 5' GAGACGACTAGTGGTGGCGGTGGCTCTCCATTC GTTTGTGAATATCAA 3' '(36 )2 G-3 5' TTACTAGCTAGCATAATAACGGAATACCCAAAA GAACTGG 3' (37)3 LAC-F 5' TATGCTAGCTAGTAACACGACAGGTTTCCCGAC TGG 3' .*l0 (38)4 LAC-B 5' ACCGAGCTCGAATTCGTAATCATGGTC 3' (39)5 LAC-B' 5' AGCTGTTGAATTCGTGAAATTGTTATCCGCT 3' F Forward Primer B Backward Primer "1 From 5' to Spe I restriction site sequence is single underlined; the overlapping sequence with the 5' end of cpIII is double underlined 2 From 5' to Nhe I restriction site sequence is single underlined; the overlapping sequence with 3' end of cpIII is double underlined.

3 From 5' to overlapping sequence with the 3' end of cpIII is double underlined; Nhe I restriction sequence begins with the nucleotide residue at position 4 and extends 5 more residues GCTAGC.

4 EcoR I restriction site sequence is single underlined.

Alternative backwards primer for amplifying LacZ; EcoR I restriction site sequence is single underlined.

2. Isolation of HIV-l-Specific Monoclonal Antibodies Produced from the Dicistronic Expression Vector, pComb2-3 In practicing this invention, the heavy (Fd 77 consisting of VH and CH1) and light (kappa) chains (VL, CL) of antibodies are first targeted to the periplasm of E. coli for the assembly of heterodimeric Fab molecules. In order to obtain expression of antibody Fab libraries on a phage surface, the nucleotide.residue sequences encoding -either the Fd or light chains must be operatively linked to the nucleotide residue sequence encoding a filamentous bacteriophage coat protein membrane anchor. A coat protein for use in this invention in providing a membrane anchor is III (cpIII or cp3). In the Examples described herein, methods for operatively linking a nucleotide residue sequence encoding a Fd chain to a cpIII membrane anchor in a fusion protein of this invention are described.

In a phagemid vector, a first and second cistron consisting of translatable DNA sequences are operatively linked to form a dicistronic DNA 20 molecule. Each cistron in the dicistronic DNA .molecule is linked to DNA expression control sequences for the coordinate expression of a fusion protein, Fd-cpIII, and a kappa light chain.

The first cistron encodes a:periplasmic secretion signal (pelB leader) operatively linked to the fusion protein, Fd-cpIII. The second cistron encodes a second pelB leader operatively linked to a kappa light chain. The presence of the pelB leader facilitates the coordinated but separate secretion of both the fusion protein and light chain from the bacterial cytoplasm into the periplasmic space.

In this process, the phagemid expression vector carries an ampicillin selectable resistance marker gene (beta lactamase or bla) in addition to the Fd-cpIII fusion and the kappa chain. The fl phage origin of replication facilitates the generation of single stranded phagemid. The 78 isopropyl thiogalactopyranoside (IPTG) induced expression of 'a dicistronic message encoding the Fd-cpIII fusion (VH, CHI, cpIII) and the light chain (VL, CL) leads to the formation of heavy and light chains. Each chain is delivered to the periplasmic -space by the pelB leader sequence, which is subsequently cleaved. The heavy chain is anchored in the membrane by the cpIII membrane anchor domain while the light chain is secreted into the periplasm. The heavy chain in the presence of light chain assembles to form Fab molecules. This same result can be achieved if, in the alternative, the light chain is anchored in the membrane via a light chain fusion protein haying a membrane anchor S 15 and heavy chain is secreted via a pelB leader into the periplasm.

With subsequent infection of E. coli with a helper phage, as the assembly of the filamentous bacteriophage progresses, the coat protein III is incorporated on the tail of the bacteriophage.. a. Preparation of Lymphocyte RNA Five milliliters of bone marrow was removed by aspiration from HIV-I asymptomatic seropositive individuals. Total cellular RNA was prepared from the bone marrow lymphocytes as described above using the RNA preparation methods described by Chomczynski et al., Anal Biochem., 162:156-159 (1987) and using the RNA isolation kit (Stratagene) according to the manufacturer's instructions. Briefly, for immediate homogenization of the cells in the isolated bone marrow, 10 ml of a denaturing solution containing M guanidinium isothiocyanate containing 71 Al of beta-mercaptoethanol was admixed to the isolated bone marrow. One ml of sodium acetate at a concentration of 2 M at pH 4.0 was then admixed with the homogenized cells. One ml of phenol that 79 had been previously saturated with H 2 0 was also admixed to the denaturing solution containing the homogenized spleen. Two ml of a chloroform:isoamyl alcohol (24:1 v/v) mixture was added to this homogenate. The homogenate was mixed vigorously for ten seconds and maintained on ice for -minutes. The homogenate was then transferred to a thick-walled 50 ml polypropylene centrifuged tube.

(Fisher Scientific Company, Pittsburgh, PA). The solution was centrifuged at 10,000 x g for minutes .at 4 0 C. The upper RNA-containing aqueous layer was transferred to a fresh 50 ml polypropylene centrifuge tube and mixed with an equal volume of isopropyl alcohol. This solution was maintained at -200C for at least one hour to i precipitate the RNA. The solution containing the precipitated RNA was centrifuged at 10,000 x g for twenty minutes at 4°C. The pelleted total cellular RNA was collected and dissolved in 3 ml of the denaturing solution described above. Three ml of Sisopropyl alcohol was added to the re-suspended total cellular RNA and vigorously mixed. This solution was maintained at -20 0 C for at least 1 *hour to precipitate the RNA. The solution containing the precipitated RNA was centrifuged at 10,000 x g for ten minutes at 4 0 C. The pelleted RNA was washed once with a solution containing ethanol. The pelleted RNA was dried under vacuum for 15 minutes and then re-suspended in dimethyl pyrocarbonate-treated (DEPC-H 2 0) H 2 0.

Messenger RNA (mRNA) enriched for sequences containing long poly A tracts was prepared from the total cellular RNA using methods described in Molecular Cloning: A Laboratory Manual, Maniatis et al., eds., Cold Spring Harbor, NY, (1982).

Briefly, one half of the total RNA isolated from a single donor prepared as described above was resuspended in one ml of DEPC-H 2 0 and maintained at I~,l ~rr 80 0 C for five minutes. One ml of 2X high salt loading buffer consisting of 100 mM Tris-HCl, 1 M NaCI, 2.0 mM EDTA at pH 7.5, and 0.2% SDS was admixed to the resuspended RNA and the mixture allowed to cool to room temperature.

The total purified mRNA was then used in PCR amplification reactions as described in Example 2c.

Alternatively, the mRNA was further purified to.

poly A+ RNA by the following procedure. The total MRNA was applied to an oligo-dT (Collaborative Research Type 2 or Type 3) column that was previously prepared by washing the oligo-dT with a solution containing 0.1 M sodium hydroxide and 5 mM EDTA and then equilibrating the column with 15 DEPC-H 2 0. The eluate was collected in a sterile polypropylene tube and reapplied to the same column after heating the eluate for 5 minutes at 65 0

C.

The oligo-dT column was then washed with 2 ml of high salt loading buffer consisting of 50 mM 0 Tris-HCl at pH 7.5, 500 mM sodium chloride, 1 mM EDTA at pH 7.5 and 0.1% SDS. The oligo dT column was then washed with 2 ml of IX medium salt buffer consisting of 50 mM Tris-HCl, pH 7.5, 100 mM, 1 mM EDTA and 0.1% SDS. The messenger RNA was eluted from the oligo-dT column with 1 ml of buffer consisting of 10 mM Tris-HCl at pH 7.5, 1 mM EDTA at pH 7.5, and 0.05% SDS. The messenger RNA was purified by extracting Ehis solution with phenol/chloroform followed by a single extraction with 100% chloroform. The messenger RNA was concentrated by ethanol precipitation and resuspended in DEPC The resultant purified mRNA contained a plurality of anti-HIV encoding V H and VL sequences for preparation of an anti-HIV-1 Fab DNA library.

81 b. Construction of a Combinatorial HIV-1 Antibody Library 1) Selection of Oliconucleotide Primers The nucleotide sequences encoding the immunoglobulin protein CDR's are highly variable. However, there are several regions of conserved sequences that flank the V region domains of either the light or heavy chain, for instance, and that contain substantially conserved nucleotide sequences, sequences that will hybridize to the same primer sequence. Therefore, polynucleotide synthesis (amplification) primers that hybridize to the conserved sequences and incorporate restriction sites into the DNA homolog produced that are suitable for operatively linking the synthesized DNA fragments to a vector were constructed. More specifically, the primers were Sdesigned so that the resulting DNA homologs produced can be inserted into .an expression vector 20 of this invention in reading frame with the upstream translatable DNA sequence at the region of the vector containing the directional ligation means.

For amplification of the VH domains, primers were designed to introduce cohesive termini compatible with directional ligation into the unique Xho I and Spe I sites of the pComb2-3 expression vector. In all cases, the 5' primers VHIa CAGGTGCAGCTCGAGCAGTCTGGG 3' SEQ ID NO 42) and VH3a GAGGTGCAGCTCGAGGAGTCTGGG 3' SEQ ID NO 43) were designed to maximize homology with the VHl and VH 3 subgroup families, respectively, although considerable cross-priming of other subgroups was expected. The Xho I restriction site for cloning into the pComb2-3 vector is underlined. The 3' primer CGlz having the nucleotide sequence GCATGTACTAGTTTTGTCACAAGATTTGGG 3' (SEQ ID NO 44) used in conjunction with the 5' primers is the 82 primer for the heavy chain corresponding to part of the hinge region. The Spe I site for cloning into the pComb2-3 vector is underlined.

The nucleotide sequences encoding the V L domain are highly variable. However, there are several regions of conserved sequences that flank the V

L

domains including the JL, VL framework regions and

V

L leader/promotor. Therefore, amplification primers were constructed that hybridized to the I conserved sequences and incorporate restriction sites that allow cloning the amplified fragments into the pComb2-3 expression vector cut with Sac I and Xba I.

For amplification of the kappa V L domains .15 analogous to the heavy chain primers listed above, the 5' primers, VKIa GACATCGAGCTCACCCAGTCTCCA 3' SEQ ID NO 45) and VK3a GAAATTGAGCTCACGCAGTCTCCA 3' SEQ ID NO 46), were used. These primers also introduced a Sac I restriction endonuclease site indicated by the underlined nucleotides to allow the V L DNA homolog I4 to be cloned into the pComb2-3 expression vector.

The 3' V L amplification primer, CKla having a nucleotide sequence

GCGCCGTCTAGAACTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCA

AG 3' (SEQ ID NO 47) corresponding to the 3' end of the light chain was used to amplify the light chain while incorporating-the underlined Xba I restriction endonuclease site required to insert the V L DNA homolog into the pComb2-3 expression vector.

All primers and synthetic polynucleotides described herein, were either purchased from Research Genetics in Huntsville, Alabama or synthesized on an Applied Biosystems DNA synthesizer, model 381A, using the manufacturer's instruction.

"V

83 2) PCR Amplification of VH and VL DNA Homologs In preparation for PCR amplification, mRNA prepared above was used as a template for cDNA synthesis by a primer extension reaction. First, 20-50 ig of total mRNA in water -was first hybridized (annealed) at 70 0 C for minutes with 600 ng (60.0 pmol) of either the heavy or light chain 3' primers listed above.

Subsequently, the hybridized admixture was used in a typical 50 il reverse transcription reaction containing 200 AM each of dATP, dCTP, dGTP and dTTP, 40 mM Tris-HCl at pH 8.0, 8 mM MgCl 2 50 mM NaC1, 2 mM spermidine and 600 units of reverse transcriptase (SuperScript, BRL). The reaction admixture was then maintained for one hour at 37 0 C :i to form an RNA-cDNA admixture.

Three il of the resultant RNA-cDNA admixture was then used in PCR amplification in a reaction.

20 volume of 100 p1 containing a.mixture of all four dNTPs at a concentration of 60 AM, 50 mM KC1, 10 mM Tris-HCl at pH 8.3, 15 mM MgC1 2 0.1% gelatin and units of Thermus aquaticus (Taq) DNA polymerase (Perkin-Elmer-Cetus, Emeryville, California) and 2:'25 60 pmol of the appropriate 5' and 3' primers listed above. The separate reaction admixtures were overlaid with mineral oil and subjected to cycles of amplification: Each amplification cycle included dtnaturation at 91 0 C for 1 minute, annealing at 52 0 C for 2 minutes and polynucleotide synthesis by primer extension (elongation) at 72 0

C

for 1.5 minutes, followed by a final maintenance period of 10 minutes at 72 0 C. An aliquot of the reaction admixtures were then separately electrophoresed on a 2% agarose gel. After successful amplification as determined by gel electrophoretic migration, the remainder of the RNA-cDNA was amplified after which the PCR products lil.-iii 84 of a common 3' primer were pooled into separate VH-and VL-coding DNA homolog-containing samples and were then extracted twice with phenol/chloroform, once with chloroform, ethanol precipitated and were .stored at -70 0 C in 10 mM Tris-HC1 at pH 7.5, and 1 -mM EDTA.

3) Insertion of V H and VL-Coding DNA Homolocs into pComb2-3 Expression 1: O Vector The VH-coding DNA homologs (heavy chain) prepared above were then digested with an excess of Xho I and Spe I for subsequent ligation.

into a similarly digested and linearized pComb2-3 in a total volume of 150 Al with 10 units of ligase at 16 0 .C overnight. The construction of the library was performed as described by Burton et al., Proc.

.Natl. Acad. Sci., USA, 88:10134-10137 (1991).

Briefly, following ligation, the pComb2-3 vector 20 •containing heavy chain DNA was then transformed by electroporation into 300 il of XL1-Blue cells.

After transformation and culturing, library size was determined by plating aliquots of the culture.

Typically the library had about 10 7 members. An overnight culture was then prepared from which phagemid DNA containing the heavy chain library was prepared.

For the cloning of the VL-coding DNA homologs (light chain), 10 Ag of phagemid DNA containing the heavy chain library was then digested with Sac I and SbaI. The resulting linearized vector was treated with phosphatase and purified by agarose gel electrophoresis. The desired fragment, 4.7 kb in length, was excised from the gel. Ligation of this vector with prepared light chain PCR DNA proceeded as described above for heavy chain. A library of approximately 107 members having heavy chain fragments operatively linked to the cpIII ntz Z 85 anchor sequence (Fd-cpIII) and light chain fragments was thus produced.

4) Preparation of Phage Expressing Fab Heterodimers Following transformation of the -resultant library produced above into XL1-Blue cells, phage were prepared to allow for isolation of HIV-1 specific Fabs by panning on target antigens. To isolate phage on which heterodimer expression has been induced, 3 ml of SOC medium (SOC was prepared by admixture of 20 g bacto-tryptone, 5 g yeast extract and 0.5 g NaCi in one liter of water, adjusting the pH to 7.5 and admixing 20 ml of ;glucose just before use to induce the expression of the Fd-cpIII and light chain heterodimer) was admixed and the culture was shaken .at 220 rpm for one hour at 370C, after which time ml of SB (SB was prepared by admixing 30 g 20 tryptone, 20 g yeast extract, and 10 g Mops buffer per liter with pH adjusted to 7) containing g/ml carbenicillin and 10 ig/ml tetracycline and the admixture was shaken at 300 rpm for an additional hour. This resultant admixture was "25 admixed to 100 ml SB containing 50 ig/ml carbenicillin and 10 jg/ml tetracycline and shaken for one hour, after which time helper phage VCSM13 (1012 pfu) were admixed and the admixture was shaken for an additional two hours. After this time, yg/ml kanamycin was admixed and maintained at 30 0

°C

overnight. The lower temperature resulted in better heterodimer incorporation on the surface of the phage. The supernatant was cleared by.

centrifugation (4000 rpm for 15 minutes in a rotor at 4 0 Phage were precipitated by admixture of 4% polyethylene glycol 8000 and 3% NaCI and maintained on ice for 30 minutes, followed by centrifugation (9000 rpm for 20 minutes 86 in a JA10 rotor at 4 0 Phage pellets were resuspended in 2 ml of PBS and microcentrifuged for three minutes to pellet debris, transferred to fresh tubes and stored at -20 0 C for subsequent screening as described below.

For determining the titering colony forming units (cfu), phage (packaged phagemid) were diluted in SB and 1 pl was used to infect 50 pl of fresh (OD600 1) XL1-Blue cells grown in SB containing 10 Ag/ml tetracycline. Phage and cells were maintained at room temperature for 15 minutes and then directly plated on LB/carbenicillin plates.

5) Selection of Anti-HIV-1 Heterodimers on Phace Surfaces Multiple Pannings of the Phaqe Library The phage library produced in Example 2b4) was panned against recombinant 20 of HIV-1 strain IIIb as described herein on coated microtiter plate to select for 'heterodimers. A second phage library was panned against recombinant gp41 (American Biotechnologies, Boston, MA) as described below to select for antigp41 heterodimers.

The panning procedure used was a modification of that originally described by Parmley and Smith (Parmley-et al., Gene, 73:305-318 (1988). Four rounds of panning were performed to enrich for specific antigen-binding clones. For this procedure, four wells of a microtiter plate (Costar 3690) were coated overnight at 40C with 25 pl of Ag/ml gpl20 or gp41 (American Biotechnologies) prepared above in 0.1 M bicarbonate, pH 8.6. The wells were washed twice with water and blocked by completely filling the well with 3% BSA in PBS and maintaining the plate at 37 0 C for one hour.

After the blocking solution was shaken out, 50 pl S<>VV 87 of the phage library prepared above (typically 1011 cfu) were admixed to each well, and the plate was maintained for two hours at 37°C- Phage were removed and the plate was washed once with water. Each well was then washed ten times with TBS/Tween (50 mM Tris-HCl at pH 7.5, 150 -mM NaCl, 0.5% Tween 20) over a period of one hour at room temperature where the washing consisted of pipetting up and down to wash the well, each time 10 allowing the well to remain completely filled with TBS/Tween between washings. The plate was washed once more with distilled water and adherent phage :ee were eluted by the addition of 50 pl of elution buffer (0.1 M HCl, adjusted to pH 2.2 with solid glycine, containing 1 mg/ml BSA) to each well followed by maintenance at room temperature for minutes. The elution buffer was pipetted up and down several times, removed, and neutralized with 3 Axl of 2 M Tris base per 50 fl of elution buffer 20 used.

Eluted phage were used to infect 2 ml of fresh

(OD

600 1) E. coli XL1-Blue cells for 15 minutes at room temperature, after which time 10 ml of SB containing 20 ig/ml carbenicillin and 10 Ag/ml tetracycline was admixed. Aliquots of 20, 10, and 1/10 il were removed from the culture for plating to determine the number of phage (packaged phagemids) that were eluted from the plate. The culture was shaken for one hour at 37°C, after which it was added to 100 ml of SB containing Ag/ml carbenicillin and 10 ig/ml tetracycline and shaken for one hour. Helper phage VCSM13 (1012 pfu) were then added and the culture was shaken for an additional two hours. After this time, 70 Ag/ml kanamycin was added and the culture was incubated at 37°C overnight. Phage preparation and further panning were repeated as described above.

88 Following each round of panning, the percentage yield of phage were determined, where yield (number of phage eluted/number of phage applied) X 100. The initial phage input ratio was .determined by titering on selective plates to be "approximately 1011 cfu for each round of panning.

The final phage output ratio was determined by infecting two ml of logarithmic phase XL1-Blue cells as described above and plating aliquots on selective plates. In the first panning for reactive phage, 4.6 X 1011 phage were applied to four wells and 7.7 X 10 5 phage were eluted. After the fourth panning 1.0 X 108 phage were eluted.

From this procedure, 20 clones were selected from the Fab library for their ability to bind to glycosylated recombinant gpl20 from the IIIB strain of HIV-1. Five clones were selected from the Fab library specific for binding to gp41. The panned phage surface libraries were then converted into 20 ones expressing soluble Fab fragments for further screening by ELISA as described below.

In addition to panning. on gpl20 of strain IIIB and gp41, also contemplated as antigens for panning of combinatorial libraries.is recombinant (IIIB strain) produced in baculovirus and recombinant gpl20 (SF2 strain) produced in Chinese Hamster Ovary cells obtained as described by Steimer et al., Science, 254:105-108 (1991).

Another antigen, a synthetic cyclic peptide, N=CH-

(CH

2 3

CO[SISGPGRAFYTG]NCH

2 CO-Cys-NH 2 (SEQ ID NO 48) prepared as described by Satterthwait et al., Bulletin of the World Health Oroanization, 68: Suppl., 17-25 (1990) corresponding to the central most conserved part of the V3 loop of gpl20 was coupled to maleimide-activated BSA. The library was panned using 1, 2 or 4 ELISA wells coated with 1 Ag of protein antigen or 10 Ag BSA-peptide per well. Four rounds of panning were carried out for 89 each antigen as described above. Eluted phage from the final round were used to infect XL1-Blue cells.

Four rounds of panning against the four antigens produced an amplification in eluted phage of between 100 and 1000 fold. The panned phage surface libraries were then converted into ones -expressing soluble Fab fragments for further screening by ELISA as described below.

10 6) Preparation of Soluble Heterodimers and Characterization of Binding Specificity to HIV-1 Antigens In order to further characterize the specificity of the mutagenized heterodimers :15 expressed on the surface of phage as described above, soluble Fab heterodimers from acid eluted phage were prepared and analyzed in ELISA assays on HIV-1 derived antigen-coated plates and by competitive

ELISA.

:20 To prepare soluble heterodimers, phagemid

DNA

from the. 20 gpl20 positive clones and the 5 gp41 positive clones prepared above was isolated and digested with Spe I and Nhe I. Digestion with these enzymes produced compatible cohesive ends.

The 4.7 kb DNA fragment lacking the gene III portion was gel-purified agarose) and self-ligated. Transformation of E. coli XL1-Blue afforded the isolation of recombinants lacking the cpIII fragment. Clones were examined for removal of the cpIII fragment by Xho I Xba I digestion, which should yield an 1.6-kb fragment. Clones were grown in 100 ml SB containing 50 Ag/ml carbenicillin and 20 mM MgC1 2 at 37 0 C until an OD 600 of 0.2 was achieved. IPTG (1 mM) was added and the culture .grown overnight at 30 0 C (growth at 37 0

C

provides only a light reduction in heterodimer yield). Cells were pelleted by centrifugation at 4000 rpm for 15 minutes in a JAlO rotor at 4 0

C.

90 Cells were resuspended in 4 ml PBS containing 34 jg/ml phenylmethylsulfonyl fluoride (PMSF) and lysed by sonication on ice (2-4 minutes at duty). Debris was pelleted by centrifugation at 14,000 rpm in a JA20 rotor at 4°C for 15 minutes. The supernatant was used directly for ELISA analysis as described below and was stored at For the study of a large number of clones, ml cultures provided sufficient heterodimer for analysis. In this case, sonications were performed in 2 ml of buffer.

Assays as described above were also performed for the gp41-specific clones.

a) Screening by ELISA /!ii The soluble heterodimers prepared above were assayed by ELISA. For this assay, gpl20 and gp41 were separately admixed to individual wells of a microtiter plate as described above for the panning procedure and maintained at 4°C overnight to allow the protein solution to adhere to the walls of the well. After the maintenance period, the wells were washed five times with water and thereafter maintained for one hour at 37°C with 100 yl solution of 1% BSA diluted in PBS to block nonspecific sites on the wells.

Afterwards, the plates were inverted and shaken to remove the BSA solution. Twenty-five pil of soluble heterodimers prepared above reactive with the specific glycoprotein substrate were then admixed to each well and maintained at 37°C for one hour to form immunoreaction products. Following the maintenance period, the wells were washed ten times with water to remove unbound soluble antibody and then maintained with a 25 yl of a 1:1000 dilution of secondary goat anti-human IgG F(ab') 2 conjugated to alkaline phosphatase diluted in PBS conitaining 1% BSA. The wells were maintained at 37°C for one V. t&ftc~44 91 hour after which the wells were washed ten times with water followed by development with 50 pl of p-nitrophenyl phosphate (PNPP). Color development was monitored at 405 nm. Positive clones gave A405 values of.>1 (mostly .after 10 minutes, whereas negative clones gave values of 0.1 to 0.2.

Approximate concentrations of Fab were determined by ELISA using a sandwich ELISA as described by Zebedee et al., Proc. Natl. Acad.

10 Sci., USA,.89:3175-317 9 (1992) and are presented in the first column of Figure 6. In addition, since :F abs are expressed in E. coli and the fraction of Scorrectly assemble protein can vary, the amount of Fab reacting with gpl20 was also assessed by ELISA titration. That data is also presented in Figure 6 in the second column.

For the clones panned against the HIV-1 derived antigens,. after conversion of the panned phage surface libraries to ones expressing soluble 20 Fab fragments, 30-40 colonies were used to transform XL1-Blue cells and the supernates screened in ELISA assays against the antigen used in panning. Generally greater than 80% of the supernates tested positive. A representative number of positives were then selected from each antigen panning for further analysis.

Competitive ELISA with Soluble and CD4 Immunoreactive heterodimers as determined in the above ELISA were then analyzed by competition ELISA to determine the affinity of the selected heterodimers. The ELISA was performed as described above on microtiter wells separately coated with 5 ig/ml of gpl20 or soluble CD4 (American Biotechnologies) in 0.1 M bicarbonate buffer at pH 8.6. Increasing concentrations of soluble or free gpl20 ranging in concentration from 92 11 M up to 10- 7 M diluted in 0.5% BSA/0.025% Tween were admixed with soluble heterodimers, the dilutions of which were determined in titration experiments that resulted in substantial reduction of OD values after a 2-fold dilution. For the CD4 competition assays, increasing concentrations of soluble or free CD4 ranging in concentration from 11 M up to 10.6 M diluted in 0.5% BSA/0.025% Tween were admixed with soluble heterodimers. The 1 0 plates were maintained for 90-120 minutes at 37°C and carefully washed ten times with 0.05% Tween o 20/PBS before admixture of alkaline phosphatase-labelled goat anti-human IgG F(ab')2 at a dilution of 1:500 followed by maintenance for 1 hour at 37°C. Development was performed as described for ELISA.- To establish the relationship between neutralizing ability as described.in Example 3 below could be related to antigen binding affinity °of HIV-l-specific Fabs, competition ELISAs were carried out where soluble gpl20 was competed with gpl20 coated on ELISA plates for Fab binding.

Figure 7 shows that all Fabs were competed from binding to gpl20 with a IC 50 of approximately 10 9

M

free gpl20. In addition as shown in Example 3, there is no correlation between antigen affinity and neutralization. The Fabs tested included Fabs 4, 12, 21 and 7 that are members of the same groups as determined by sequence analysis and comparison as described in Example 9. Fabs 13, 27, 6, 29, 2 and 3 are all members of the different groups as determined by sequence analysis and comparison as described in Example 9. Loop 2 is an Fab fragment selected from the same library as the other Fabs but which recognizes the V3 loop. Only with the V3 loop peptide was competition carried out with from the SF2 strain.

93 To investigate whether neutralization could be associated with blocking of the gpl20-CD4 interaction, competition ELISAs were carried out with soluble CD4 competing with Fabs for binding to gpl20-coated ELISA wells. The results are shown in Figure 8. P4D10 and loop 2 are controls not -expected to be competed by CD4. P4D10 is a mouse monoclonal antibody reacting with the V3 loop of (IIIB). Loop 2 Fab competition was carried out using gpl20 (SF2). As shown in Figure 8 the binding of all Fabs with the exception of the controls was inhibited with an IC 50 of approximately 8 M of soluble CD4. In addition, no difference was detected between the neutralizing and non-neutralizing Fabs to gpl20 inhibited by CD4.

This implies that blocking of the CD4-gpl20 9 interaction is unlikely to be an important factor in Fab neutralization of the HIV-1 virus.

Similar competition assays were performed with the Fabs panned against the four HIV-1 derived antigens. The 19 Fabs derived from panning against (IIIB) showed apparent affinities (1/concentration at 50% inhibition) for (IIIB) in the range 10 7 10 9 M with most being 1-3 X 10" 8 M. The panning procedure tends to select strongly for tight binders so a grouping into a relatively narrow band of affinities was expected.

Of 16.Fabs derived from panning against (IIIB), 6 were also reactive with gpl20 (IIIB) and competition ELISAs showed they had similar apparent affinities as the gpl20-panned Fabs. The reactive clones from the gpl60 panning showed a .lower ELISA reactivity with gpl60 and could not be satisfactorily competed with gpl60. They may be directed against gp41 but were not pursued here.

Eight Fabs derived from panning against gpl20 (SF2) also showed strong ELISA reactivity with (IIIB) and gave similar apparent binding I I i ,Y iq 94 affinities. Four Fabs were derived from panning against the V3 loop peptide. Of these Fabs, 2 reacted in ELISA with gpl20 (SF2) but none with (IIIB). The apparent binding affinity of these loop binders to gpl20 (SF2) was 10.8 M.

To complete the survey in terms of strain cross-reactivity of Fabs, those derived from the and gpl60 (IIIB) pannings were examined for ELISA reactivity with gpl20 (SF2). All were *.o0 reactive. Therefore, all the Fabs examined, with the exception of those selected by panning against the V3 loop peptide, bound to gpl20 from IIIB and SF2 strains.

The Fabs were screened for CD4 inhibition of their binding to gpi20 (IIIB) immobilized on ELISA i: Swells. All, again with the exception of the V3 loop binders, showed sensitivity to CD4 inhibition.

The inhibition constants were in the range 10" 7 to 9 M. Binding Affinity Determination Using Surface Plasmon Resonance Binding affinities were determined for six of the Fabs using surface plasmon resonance.

Surface plasmon resonance was performed as it is a more accurate method for measuring affinity than competition ELISA. The six Fabs were chosen based upon sequence analysis which revealed that the heavy chains could be organized into 7 groups (Example 9).

Each group contained members with identical V-D and D- J joining regions, implying a common clonal origin with varying numbers of differences elsewhere in the VH domain. Six Fabs were chosen as a representative of each respective group for further study as described herein. The single member of the seventh group was not included in these studies. The binding affinities of the six Fabs that are directed against the CD4 binding site of the gpl20 envelope 95 glycoprotein were determined using surface plasmon resonance as follows.

A Pharmacia BIAcore machine was used for the binding affinity determinations as previously described in Malmborg, et al., J. Immunol., 35:643- 650 (1992) and Mattsson, et al., J. Immunol. Meth., -145:229-240 (1991). Optimization for the Fab fragments involved a number of steps. Two separate channels on a biosensor chip were coated with derived from the HIV-1 strain LAI (Repligen, Cambridge MA) such that one channel could be used for the determination of on-rate constants (kon) and the other for the determination of off-rate constants (koff) For immobilization of antigen on the sensor surfaces, a flow rate of 5 jl/min of PBS, pH 7.4.

was established over the biosensor chip. The chip was then activated by injecting 30 Al of activation solution (Pharmacia Biosensor, 50% 0.2 M N-ethyl- (3-diethylaminopropyl)-carbodiimide, 50% Nhydroxysuccinimide). The flow rate was then adjusted to 10 Al/min and the gpl20 was injected in 10 mM sodium acetate buffer, pH 4.5. When association rates were to be determined, 25 il of gpl20 at 10 jg/ml was injected (a.final level of 4000 Response Units Twenty il of gpl20 at 2 Ag/ml were injected for the determination of dissociation constants (a final level of 800 RU).

In both cases; a flow.rate of 5 l/min was reestablished following the gpl20 injection and the chip was blocked from any further immobilization by the injection of 30 il of 1 M ethanolamine, pH (Pharmacia Biosensor).

For determination of on-rate constants a series of dilutions were made for each Fab to give

C

96 final concentrations in the range of 1 to 20 yg/ml.

Ail of each Fab solution was injected in separate experiments over the immobilized gpl20 at a flow rate of 5 jul/min. The change in response per unit time (dR/dt) was plotted against time for each concentration. The slopes of each of these graphs were then plotted against their corresponding concentrations to give a final graph from which the .on-rate constant could be read.

.'L0 For determination of off-rate constants (koff) Al of each Fab solution at 150 pg/ml were injected over the immobilized antigen at a flow rate of 5 il/min. Once the reaction had reached equilibrium, the Fab was removed from the antigen at a constant flow rate of 50 ji/min. A plot was then made of ln(R/R 0 against ti-to for the dissociation phase. R i is the response at time t i and R 0 is the initial response at time to. The slope of this graph was taken to be the off-rate constant. Affinities (Ka) were then calculated and expressed as kon/koff.

The apparent affinities of the panel of recombinant Fabs isolated from the donor as determined in competition ELISA and surface plasmon resonance were compared. Values of approximately 8 M-1 were obtained by competition ELISA as described in Example 2b6c in which the soluble and immobilized gpl20 competed for binding to Fab in bacterial supernatants. Such a methodology only gives-an approximate measure of affinity.

Therefore, the affinities of six of these Fabs were measured using real-time biospecific interaction analysis (surface plasmon resonance) in order to obtain more accurate affinity constant values. The results are reproducible with a standard deviation from the mean of approximately 5% as determined by calculating a number of the affinity constants in triplicate. All Fabs examined have affinities in ~tAI4~A" t.

97 the range of 5 x 107 to 1 x 108 M as determined in surface plasmon resonance (Table These values are in broad agreement with those derived from competition ELISA. These values imply no correlation between affinity for recombinant derived from LAI and the ability to neutralize the HXBc2 clone of HIV-1 derived from LAI as assessed in Example 3c.

"l0 Table 4 Fab k 0 n (M-S'S- 1 koff Ka(Ml.) Sb3 9.6 x 10 3 1.8 x 10" 4 5.1 x 1C b6 1.6 x 104 1.6 x 10- 4 9.7 x 1C bll 5.6 x 104 4.3 x 10- 4 1.3 x 1C d b12 4.5 x 104 4.3 x 10- 4 1.1 x 1C b13 1.1 x 104 1.4 x 10- 4 7.9 x 1C b14 6.0 x l04 6.5 x 10- 4 9.2 x 1C Also contemplated are competition ELISA and surface plasmon resonance assays where •the binding of HIV-1 recombinant Fabs of this invention is performed in the presence of excess Fabs of this invention as well as those HIV-1 antibodies, polyclonal or monoclonal, present in patient sera, either asymptomatic or symptomatic, or obtained by other means such as EBV transformation and the like. The ability of an exogenously admixed antibody to compete for the binding of a characterized Fab of this invention will allow for the determination of equivalent antibodies in addition to unique epitopes and binding specificities.

~4~Z 98 3. Neutralizing Activity of Recombinant Human Fab Fragments Against HIV-1 In Vitro Binding of antibodies to viruses can result in .loss of infectivity or neutralization and, although not the only defense mechanism against viruses, it is widely accepted that antibodies have an *i: important role to play. However, understanding of the molecular principles underlying antibody ~0 neutralization is limited and lags behind that of the other effector functions of antibody. Such understanding is required for the rational design of vaccines and for the most effective use of passive antibody for prophylaxis or therapy. This is particularly urgent for the human iij; immunodeficiency viruses.

A number of studies have led to the general conclusion that viruses are neutralized by more than one mechanism and the one employed will depend on factors such as the nature of the virus, the epitope recognized, the isotype of the antibody, the cell receptor used for viral entry and the virus:antibody ratio. The principle mechanisms of neutralization can be considered as aggregation of virions, inhibition of attachment of virus to cell receptor and inhibition of. events following attachment such as fusion of viral and cellular membranes and secondary uncoating of the virion.

One of the important features of the third mechanism is that it may require far less than the approximately stoichiometric amounts of antibody expected for the first' two mechanisms since occupation of a small number of critical sites on the virion may be sufficient for neutralization.

For instance it has been shown that neutralization of the influenza A virion obeys single hit kinetics as described by Outlaw et al., Epidemiol. Infect., 106:205-220 (1992).

99 Intensive studies have been carried out on antibody neutralization of HIV-1. For review, see Nara et al., FASEB 5:2437-2455 (1991). Most have focussed on a single linear epitope in the third hypervariable domain of the viral envelope glycoprotein gpl20 known as the V3 loop.

Antibodies to this loop are suggested to neutralize by inhibiting fusion of viral and cell membranes.

Binding to the loop resulting in neutralization can occur prior to virus-cell interaction or following "gpl20 binding to CD4. See, Nara, In Retroviruses of Human Aids and Related Animal Diseases, eds.

'Girard et al., pp. 138-150 (1988); Linsely et al., J. Virol., 62:3695-3702 (1988); and Skinner et al., J. Virol., 67:4195-4200 (1988). Features of the V3 loop are sequence variability within the loop [Goudsmit et al., FASEB 5:2427-2436 (1991) and Albert et al., AIDS, 4:107-112 (1990)] and sensitivity of neutralizing antibodies against the 20 loop to sequence variations outside the loop [Nara et al., FASEB 5:2437-2455 (1991); Albert et al., supra; McKeating et al., AIDS, .3:777-784 (1989); and Wahlberg et al., AIDS Res. Hum.

Retroviruses, 7:983-990 (1991) Hence anti-V3 loop antibodies are often strain specific and mutations in the loop in vivo may provide a mechanism for viral escape from antibody neutralization.

Recently considerable interest has focused on antibodies capable of blocking CD4 binding to gpl20. A number of groups have described the features of these antibodies as reacting with conformational non-linear epitopes, (b) reacting with a wide range of virus isolates and being the predominant neutralizing antibodies in humans after longer periods of infection. See, Berkower,et al., J. Virol.. 65:5983-5990 (1991); Steimer et al., Science, 254:105-108 (1991); Ho et al., J. Virol., 65:489-493 (1991); Kang et al., 4~vL~ 100 Proc. Natl. Acad. Sci., USA, 88:6171-6175 (1991); Posner et al., J. Immunol., 146:4325-4332 (1991); and.Tilley et al., Res. Virol., 142:247-259 (1991).

Neutralizing antibodies of this type would appear ,to present a promising target for potential S-therapeutics. The mechanism(s) of neutralization of these antibodies is unknown although there is some indication that this may not be blocking of virus attachment since a number of mouse monoclonal 3 antibodies inhibiting CD4 binding to gpl20 are *either non-neutralizing or only weakly neutralizing.

The generation of human monoclonal antibodies against the envelope of HIV-1 as described by :15 Burton et al., Proc. Natl. Acad. Sci., USA, 88:10134-10137 (1991) using combinatorial libraries allows a novel approach to the problem of neutralization. Given the lack of a three-dimensional structure for gpl20 and the complexity of the virus, the approach seeks to explore neutralization at the molecular level i through the behavior of related antibodies. This is possible for the following reasons: the combinatorial approach allows the rapid generation of large numbers of human antibodies; the .i antibodies (Fab fragments) are expressed in E.coli and can readily be sequenced; and antibodies have similar sequences and common structural motifs allowing functional differences to be meaningfully correlated with primary structure.

Neutralization studies were performed as described herein on the human recombinant Fab fragments from 20 clones against gpl20 prepared as described in Examples 1 and 2, all of which are strain cross-reactive and inhibited by CD4 from binding to gpl20. The results presented herein show that neutralization was not effected by virus aggregation or cross-linking of gpl20molecules on _iY~E 101.the virion surface and was not correlated with blocking of the interaction between soluble CD4 and recombinant Neutralization studies were also performed as described herein on the human recombinant Fab fragments from the gp41-reactive clones prepared as described in Examples 1 and 2. The results are presented below.

Two different assays, a p24 ELISA assay and a syncytium assay, were performed to measure neutralization ability of the recombinant human HIV-1 immunoreactive Fabs. An additional assay, a plaque assay, was performed for determining the neutralization effectiveness of the gp41-reactive Fabs. In plaque assays, CD4+ cells were cultured in the presence or absence of soluble gp41-reactive Fabs prior to inoculation with virus. Inhibition of infectivity, also referred to as neutralization, i by antibodies was expressed as the percent of 20 plaque formation in the cultures compared to cells exposed to PBS alone. Neutralization assays were also performed with an antibody molecule consisting of the light chain and the VH region of the Fab 12 and the constant regions (CH1, CH2, and CH3) of an IgGl molecule.

Quantitative infectivity microplaque and syncytial formation assays to measure neutralization were performed with the b12 IgGl and laboratory isolates MN and IIIB of HIV-1 virus. In the syncytial formation assay, virus was grown in H9 cells and infectivity measured by culturing monolayers of CEM-SS target cells with 100-200 syncytial forming units (SFUs) of virus, in the presence or absence of antibody, p24 ELISA and microplaque formation assays were also performed with primary clinical isolates of the HIV-1 virus.

In addition, the ability of the recombinant human HIV-1 immunoreactive Fabs b3, b6, b12, b13, +t 102 and b12 to neutralize the HXBc2 molecular clone of derived from HTLV-IIIB (LAI) was determined in an envelope complementation assay. The supernatant containing recombinant HIV-1 virions .from cotransfected COS-1 cells was incubated with the recombinant Fabs prior to incubation with Jurkat cells. The recombinant HIV-1 virions contained the HXBc2 clone of HIV-1 strain LAI which encodes a chloramphenicol acetyltransferase (CAT) gene. Upon infection of Jurkat cells with the recombinant HIV-1 virions, the CAT gene was expressed and CAT activity measured. Activity of the CAT gene was therefore an indication of infectivity of the Jurkat cells with the recombinant HIV-1 virion. Lack of CAT activity indicated the Jurkat cells were not infected with the recombinant HIV-1 virion. For some of these assays, the recombinant Fabs were first purified. One liter cultures of SB containing 50 pg/ml carbenicillin and 20 mM MgC12 were inoculated with appropriate clones and induced 7 hours later with 2 mM IPTG and grown overnight at 30 0 C. The cell pellets were-sonicated and the resultant supernatant were concentrated to a 50 ml .volume. The filtered supernatants were loaded on a ml protein G-anti-Fab column, washed with 120 ml buffer at a rate of 3 ml/minute and eluted with citric acid at pH 2.3. The neutralized fractions were then concentrated-and exchanged into 50 mM MES at pH 6.0 and loaded onto a 2 ml Mono-S column at a rate of 1 ml/minute. A gradient of 0-500 mM NaCl was run at 1 ml/minute with the Fab eluting in the i range of 200-250 mM NaC1. After concentrating, the Fabs were positive when titered on ELISA against gpl20 and gave a single band at 50 kD by 10-15% SDS-PAGE. Concentration was determined by absorbance measurement at 280nm using an extinction coefficient (1 mg/ml) of 1.4.

103 a. Neutralization as Measured by the p24 ELISA Assay For this assay, diluted tissue culture supernatants of HIV-1 IIIB or MN-infected peripheral blood mononuclear cells (PBMC) (50TCID 50 tissue culture infectious dose), 100 Al) were -maintained for 2 hours at 37°C with serial dilutions beginning at a dilution of 1:20, of recombinant Fab supernates prepared in Example 2b6) Control Fab supernates were also provided that included human neutralizing sera, a known human neutralizing monoclonal antibody 2F5 and the Fab fragment derived from that antibody by papain digestion, and a known mouse neutralizing monoclonal antibody and its F(ab') 2 fragment as described by Broliden et al., J. Virol. 64:936-940 (1990) PBMC (1 x 105 cells were admixed to the virus/antibody admixture and maintained for 1 hour at 37°C. Thereafter, the cells were washed and maintained in RPMI 1640 medium (GIBCO) supplemented with 10% fetal calf serum, 1% glutamine, i antibiotics and IL-2. The culture medium was changed at days 1 and 4. At 7 days post-infection, supernates were collected and analyzed by HIV-1 p24 antigen capture ELISA as described by Sundqvist et al., J. Med. Virol., 29:170-175 (1989) the disclosure of which is hereby incorporated by reference. Neutralization was defined as positive if an 80% or greater reduction of optical density at 490nm in the culture supernatant occurred as compared to negative Fab or negative human serum.

Tests with all Fabs, mAbs and sera were repeated on at least two occasions.

b. Quantitative Infectivity Assay Based on Syncvtial Formation A quantitative neutralization assay with the MN strain of HIV-1 was performed as described ri&sS^idifttoa^^^s^ *~lSa 104 by Nara et al., AIDS Res. Human Retroviruses, 3:283-302 (1987), the disclosure of-which is hereby incorporated by reference. Monolayers of CEM-SS target cells were cultured with virus, in the .presence or absence of antibody, and the number of -syncytia forming units determined 3-5 days later.

An equivalent amount of virus was used in the assays to allow direct comparison of the various antibody concentrations tested. The assays were repeatable over a virus-surviving fraction range of 1 to 0.001 within a 2 to 4-fold difference in the concentration of antibody (P<0.001).

i

I

1: i i..

c. Neutralization as Measured by the Envelope Complementation Assay The ability of purified recombinant Fabs b3, b6, bll, b12, bl3, and b14 to neutralize the HXBc2 gpl20 molecular clone of the HIV-1 (LAI) isolate was assessed in an envelope complementation assay (Helseth et al., J. Virol., 65:2119-2123 (1991)). Briefly, COS-1 cells were cotransfected with a plasmid expressing envelope glycoprotein 120 derived from HIV-1 (LAI) and a plasmid containing an env-defective HXBc2 clone and encoding the bacterial CAT gene. Equal fractions of the cell supernatants containing recombinant virions were incubated at 37 0 C for 1 hour with varying concentrations of recombinant Fab (0.1 20 ig/ml) or control monoclonal antibody 110.4 prior to incubation with Jurkat cells. Three days postinfection, the Jurkat cells were lysed and CAT activity measured. Neutralization was expressed as a decrease in the percentage of residual chloramphenicol transferase (CAT) activity.

Control monoclonal antibody 110.4 is a strongly neutralizing antibody directed to the V3 loop of the HXBc2 HIV-1 strain.

i! 1:i 105 d. Results of the Neutralization Assays for cpl120 Assays were generally repeated at least i twice with reproducible results. For the data reported in Figure 6, the gpl20-specific Fab supernates were divided into two parts, one being -used in the p24 assay and the other in the syncytia assay. A dash indicates that there was no neutralization at 1:20 dilution in the p24 assay 10 and 1:16 in the syncytial assay (with most clones showing.no detectable neutralization at a 1:4 dilution). Neutralization titers are indicated in the figure. For the p24 assay, the titer corresponds to the greatest dilution producing reduction in absorbance in ELISA. For the syncytia assay, Fabs 4 and 12 produced >95% neutralization at a 1:4 dilution of supernate and 80 and reduction at 1:128 dilution respectively. These Fabs were effective neutralizers in both types of 20 assays. They have also been shown to neutralize infection by IIIB and RF strains using a PCR-based assay of proviral integration. Fabs 6 and 7 showed no neutralization in the syncytia assay but other supernate preparations showed activity. Fab 13 was consistently effective in the p24 assay but not in the syncytia assay. A number of other clones show lower levels of neutralizing ability.

Fabs were purified from a selection of some of the clones as described above and used in both neutralization assays. As shown in Figure 9, Fabs 4 and 12 were again effective in both assays at similar levels with for example 50% inhibition of syncytial formation at an Fab concentration of approximately 20 nM (1 The results shown are derived from the syncytia assay using the MN strain. Fabs 7 and 21 were equally effective in the syncytial assay but somewhat less so in the p24 assay. The p24 assay indicated greater than ii; r ,n 106 neutralization of HIV-1 MN strain for Fab 4 at 3, Fab 7 at 15, Fab 12 at 3, Fab 13 at 4 and Fab 21 at 7 pg/ml, respectively. Fab 13 however was ineffective in the syncytial assay at 25 pg/ml.

For the IIIB strain, greater than 80% -neutralization was observed for Fab 4 at 13, Fab 7 at 15, Fab 12 at 7 and Fab 21 at 14 Jg/ml, respectively. Although Fab 11 was not effective in neutralization assays when unpurified as shown in .aO Figure 6, following purification, Fab 11 was Sequally effective as Fab 12 in neutralizing HIV-1.

For this reason, the Fab is being deposited with the ATCC as described in Example 12 along with Fab 12 and Fab 13..

The ability of purified recombinant Fabs b3, b6, bll, b12, b13, and b14 to neutralize the HXBc2 gpl20 molecular clone of the HIV-1 (LAI) isolate was assessed in an envelope complementation assay. Figure 23 shows the concentration dependence of Fab 0 neutralization of the.HXBc2 clone in this assay.

All of the Fabs neutralize effectively at the I highest concentration measured (20 Ag/ml).

Irrelevant Fabs, Fabs directed to surface glycoproteins on other viruses such as RSV, do not neutralize in this assay. Examination of the lower i-; concentrations clearly reveals that Fab b12 is the most effective neutralizer. The neutralizing potency of Fab b12 was greater than that of the 110.4 whole monoclonal antibody tested in parallel.

The 110.4 antibody is one of the most potent antibodies directed against the V3 loop of the HXBc2 HIV-1 strain (Thali, M. and J. .Sodroski, unpublished observations). In other studies, Fab b12 has been found to show exceptional neutralizing ability towards laboratory (Example 3 and Barbas et al., Proc. Natl. Acad. Sci., USA, 91, in press (1994)) and field isolates of HIV-1 as described in Example Ir 107 There are a number of conclusions arising from the data shown in the Figures 6, 9 and 23. It is apparent that HIV-I can be neutralized without virion aggregation or cross-linking of molecules on the virion surface since monovalent Fab fragments are effective. To further confirm -this finding, a Fab fragment was produced by papain digestion of a known neutralizing human monoclonal antibody. As shown in Figure 6, the Fab fragment was approximately equally effective as the whole IgG in neutralization of the MN strain of HIV-1.

This is consistent with results on Fabs prepared from two mouse monoclonal antibodies to the V3 loop. An F(ab') 2 fragment of a mouse monoclonal antibody was somewhat less effective than the parent IgG in neutralization of the MN strain.

Interestingly, the fragments from these control antibodies were relatively poor in neutralizing the IIIB strain of HIV-l. The results also show that 20 there appears to be a difference between the two assays employed since Fab 13 was consistently effective in one assay but not the other. The principal variables were the incubation time of the virus and antibody prior to infection (2 hours for the p24 assay and 0.5 hours for the syncytial assay), the amount of virus used for infection, the cells used to propagate virus (human PBMCs for the former and H9 cells for the latter) and the cells infected (human PBMCs for the former and CEM.SS cells for the latter). Of these, there is a strong .possibility that the MN virus used in the two assays, having been passaged through different cells, is critically different.

The Fabs show a spectrum of neutralizing ability for gpl20 from a molecular clone HXBc2 derived from the HIV-l strain LAI in the envelope complementation assay. Fab b12 exhibited the greatest potency of neutralization and was even 108 more effective in this assay than a whole antibody directed to the V3 loop of gpl20. Neutralizing ability is not correlated with either the apparent affinity of the Fab for gpl20 derived from the .recombinant HIV-1 strain LAI as estimated by competition ELISA or the affinity for gpi20 derived from HIV-l strain LAI as determined by surface plasmon resonance. For example, Fabs b6, b12, and b14 have very similar affinities by surface plasmon 0 resonance (Table 4) but different neutralization ability in the envelope complementation assay (Figure 23). Similarly, neutralization is not correlated with the ability of the Fab to compete with soluble CD4 in a competition ELISA.

15 e. Results of the Neutralization Assays for 9. crp4l The gp41-reactive Fabs exhibited specificity to the conformation epitope of gp41 20 including amino acid residues in positions 565-585 and 644-663. The five selected gp41-specific Fabs were designated DL 41 19, DO 41 11, GL 41 1, MT 41 12 and SS 41 8. Neutralization assays were performed as described above for the Fabs. In the plaque assays, the data shown is the concentration of Fab in micrograms/milliliter required to achieve 50%1 of neutralization. The data for the other two neutralization assays is also expressed in micrograms/milliliter of Fab required to neutralize infection as defined in the description. of the p24 and syncytial assays above.

The results of the three neutralization assays, plaque, syncytial and p24, are presented in Table The MN and IIIB HIV strains were used as indicated in Table 5 for the assays. The abbreviation "ND" stands for not determined when indicated in the table.

109 Table Assay/Strain Plaque Syncytial P24 Fab MN IIIB IIIB MN IIIB DL 41 19 <4 <40 1.4 ND ND.

DO 41 11 <40 7.1 2.3 0.9 ND -GL 41 1 <4 <4 1.7 ND MT 41 12 <40 <40 5.5 4.5 SS 41 8 <4 <4 2.2 ND 7.1 As shown in Table 5, all five Fabs were effective at neutralizing both MN and IIIB strains of HIV in either plaque, syncytial or p24 assays.

Fabs DL 41 19 and DO 41-11 exhibited strain specificity in the plaque assay where the former -was ten-fold more effective at inhibiting plaque formation with the MN strain than with the IIIB strain. The opposite specificity was seen with the DO 41 11 Fab. However, both Fabs exhibited 20 comparable neutralization as measured by the syncytial assay. Two Fabs, GL 41 1 and SS 41 8, were equally effective at inhibiting plaque formation .with either MN or IIIB strains. The Fab i.

MT 41 12 was similarly not strain-specific although neutralization required 10 .fold more antibody. No ii strain specificity was evident when Fab MT 41 12 was used in p24 assays where the same amount of antibody was equally effective. All five antibodies were neutralized IIIB as measured in the syncytial assay.

Thus, the five gp41-specific Fabs neutralized HIV-1 MN and IIIB in at least two of the three assays used for measuring neutralizing activity.

Moreover, strain specificity was prevalent in two of the five assays as measured by the plaque assay.

Based on these differential neutralization characteristics, the gp41-specific Fabs provide useful therapeutic reagents for neutralizing HIV-1.

110 4. Construction of a Mammalian Expression Vector pEel2 Combo BM 12 for the Expression of an IqGl Antibody Molecule with the Fab from b12 (b12 IqG1) Although Fab bl2 is capable of neutralizing some primary isolates, the corresponding whole antibody molecule is likely to be more effective.

The whole antibody, consisting of the Fab fragment :.410 and the Fc domain,- participates in the elimination of foreign cells by first binding specifically to the foreign cell via the Fab portion and interacting with other cells in the immune system via the Fc domain. The Fc domain also enables the antibody to bind complement.

e":b.l Fab b12 was converted to a whole IgGI molecule (b12 IgGl) by cassetting the variable heavy chain (VH) and light chain genes .into .a vector created for high-level mammalian expression, b12 IgGl used .l in the neutralization studies was prepared by expression in Chinese hamster ovary (CHO) cells and purified by affinity chromatography. V:: S"The strategy to convert the Fab b12 to a whole IgGl molecule was similar to that described previously for the generation of a whole antibody beginning with a phage derived Fab (Bender, et al., Hum. Antibod. Hybridomas, 4:74-79 (1992)). a. Construction of b12 Heavy Chain IqGl pSG- 5 Mammalian Expression Vector 1) 'Modification of b12 Heavy Chain Variable Region to Introduce a Kozak..

Sequence. Mammalian Leader Sequence, and Human VH Consensus Sequence First, the b12 VH region was cloned into a pSG-5 expression vector (Green et al., Nucl.

Acids Res., 16:369 (1988)) to fuse the bl12 VH tc the heavy chain constant domains (CH1, CH2, and r~n Aa~zZcat~4t,±L~t$ A~x~v~i~ 111 CH3) of an IgGl antibody molecule. The double-stranded Fab b12 DNA was used as a template for isolating the gene encoding the VH.region of the Fab b12, the amino acid residue sequence of which is listed in SEQ ID NO 66. Fab b12 DNA and mouse B73.2 IgG1 DNA (Whittle, et al., Protein -Enq., 1:499 (1987) and Bruggmeman, et al., J. Exp.

Med., 166:1351 (1987)) were used as templates for a PCR amplification for the construction of a DNA 10 fragment consisting of the unique Kozak sequence for the control of heavy chain expression, the mouse B72.3 heavy chain leader sequence (MEWSWVFLFFLSVTTGVHS (SEQ ID NO 155 from amino acid residue sequence 1 to the human VH consensus sequence (QVQLVQ (SEQ ID NO 155 from amino acid residue sequence 21 to and the VH region of the Fab b12. Altering the beginning of the VH from the mouse consensus sequence to the human consensus sequence also destroyed the original Xho I cloning i 20 site. The restriction sites EcoR I and Sst I were *introduced in the amplification reaction and were il located at the.5' and 3' ends of the fragment, :i o. ii. respectively. The procedure for creating the modified VH fragment by combining the products of the two separate PCR amplifications is described below.

The primer pair, HC-1 (SEQ ID NO 157) and HC-2 (SEQ ID.NO 158) as shown in Table 10, was used in the first PCR reaction to amplify a portion of the Fab b12 VH gene and incorporate the human heavy chain consensus sequence into the 5' end of the VH fragment and introduce an Sst I cloning site in the 3' end of the VH fragment. In addition, the 5' PCR primer introduces sequences into the VH fragment which form 27 base pairs of homology with the mouse leader sequence fragment prepared below. The 27 base pairs of homology in the fragments is used in a subsequent PCR reaction to fuse the two PCR -~3AW 112 products (Yon and Fried, Nucl. Acids Res., 17:4895 (1989)) to form a modified VH fragment consisting of the EcoR I cloning site, the mouse leader sequence 72.3, the human consensus sequence, the remaining VH coding sequence, and the Sst I cloning site. For the PCR reactions, 1 Al containing.100 ng of Fab b12 DNA was admixed with 10 p.1 of 10X PCR buffer in a 0.5 ml microfuge tube. To the DNA admixture, 8 Al of a 2.5 mM solution of dNTPs 10 (dATP, dCTP, dGTP, dTTP) was admixed to result in a final concentration of 200 micromolar (pM) of each dNTP. 1 Al (equivalent to 20 picomoles of the 5' forward HC-1 primer and 1 Ail (20 pM) of the 3' backward HC-2 primer were admixed into the DNA solution. To the admixture, 73 p1 of sterile water and 2.5 units of Taq DNA polymerase was added. Two drops of mineral oil were placed on top of the admixture and 35 rounds of PCR amplification in a thermocycler were performed. The amplification cycle consisted of 52°C for 1 minute, 72°C for 2 minutes and 94°C for 0.5 minutes.

The primer pair, HC-3-. (SEQ ID NO 159) and HC-4 (SEQ ID NO 160) as shown in Table 10, was used in a separate PCR reaction to amplify the mouse B72.3 leader sequence and incorporate an EcoR I cloning site at the 5' end of the fragment and to introduce a 27 base pair sequence which has homology to the modified VH fragment prepared above. Doublestranded DNA encoding the mouse B73.2 IgGl (Whittle, et al., suora) was used as a template for preparation of the mouse 72.3 leader sequence. The PCR reaction to prepare the mouse leader sequence fragment was performed using the same conditions as described above for the preparation of the modified VH fragment.

The resultant PCR modified b12 VH DNA fragment and mouse leader sequence fragment were purified by electrophoresis in a 2.5% Nu-Sieve agarose gel 113 (FMC). The area in the agarose containing the modified b12 VH DNA fragment and mouse leader sequence fragment were .excised from the agarose.

A third PCR amplification using the primer pairs, HC-1 (SEQ ID NO 157) and HC-3 (SEQ ID NO 159) as shown in Table 10, was performed to fuse the mouse leader fragment with the modified

VH

fragment. The primers used for this amplification were designed to preserve an EcoR I site, a unique 10 Kozak sequence, and the mouse B72.3 heavy chain leader sequence on the 5' end of the amplified fragment and to preserve the Sst I cloning site on the 5' end of the amplified fragment. The templates used in this PCR reaction were the two purified PCR reaction products described above.

The PCR reaction and subsequent purification of the PCR product were performed as described above. i 2) Modification of b12 Heavy Chain 20 Variable Region to Eliminate .a BqlII Restriction Site The bl2 modified heavy chain fragment prepared in Example 4al contained a Bgl II cloning site at amino acid residue 87 which would interfere with the insertion of the heavy chain fragment into the pEE6 mammalian expression vector.

The Bgl II restriction site was therefore eliminated in a PCR reaction using primers which destroyed the Bgl II restriction site while preserving the encoded amino acid, arginine at amino acid residue 87 of the modified b12 heavy chain fragment. The primer pair, HC-1 (SEQ ID NO 157) and HC-6 (SEQ ID NO 162) as shown in Table 10, was used in the first PCR reaction to preserve the 5' region of the modified b12 heavy chain fragment and destroy the Bgl II restriction site at amino acid residue 87 of the heavy chain. The HC-6 primer introduces

I.

114 sequences into the VH fragment which form 32 base pairs of homology with the remaining portion of the VH fragment which will be prepared as described below. The 32 base pairs of homology in the fragments was used in a subsequent PCR reaction to fuse the two PCR products (Yon and Fried, supra) to form a modified VH fragment as described above but without the Bgl II restriction site. The PCR reaction was performed and the PCR products were 0 purified as described in Example 4al.

The primer pair, HC-2 (SEQ ID NO 142) and (SEQ ID NO 145) as shown in Table 10, was used in the second PCR reaction to preserve the 3' region of the modified b12 heavy chain fragment and destroy the Bgl II restriction site. The primer introduces sequences into the VH fragment which form 32 base pairs of homology with the remaining portion of the VH fragment which was prepared in the first PCR reaction. PCR products which have incorporated the HC-5 and HC-6 primers contain 32. base pairs of overlapping sequences which are identical.. It is the annealing of the -two PCR products at these 32 base pairs during the subsequent PCR reaction which fuses the two portions of the VH fragment together to recreate the entire VH fragment. as described in Yon and Fried (supra).

A third PCR amplification using the primer pairs, HC-1 (SEQ ID NO 157) and HC-3 (SEQ ID NO 159) as shown in Table 10, was performed to fuse the two VH fragments in which the Bgl II restriction site had been destroyed. The primers used for this amplification were designed to preserve an EcoR I site, a unique Kozak sequence, and the mouse B72.3.heavy chain leader sequence on the 5' end of the amplified fragment and the Sst I cloning site on the 3' end of the amplified fragment. The templates used in this PCR reaction 115 were the two purified PCR reaction products described above. The PCR reaction and subsequent purification of the PCR product were performed as described in Example 4a1.

Table 1

SEQ

ID NO Primer (141)' HC-l (142)2 HC-2 (144)2 HC-4 (145) 4 HC-5 (146-)4 HC-6 (147) LC-1 (14 9)3 LC-3 (150) LC-4 5' CAGGTTCAGCTGGTTCAGTCCGGGG CT 3' 5' CCTTGGAGCTCACGATGACCGTGG

T

TCCTTGGCCCCAGACGTCC3' 5 GGCCGCGAATTCGCCGCCACCATGG

AATGGAGCTGGGTCTTTCTCTTCTT

CCTGTCAGTA 3' (B3) 5' AGCCCCGGACTGAACCAGCTGAAC C TG 3' 5' GGAGTTGAGGAGCCTCAGGTCTGCA GACACGG 3' (B3) 5' CCGTGTCTGCAGACCTGTGGCTCCT CAACTCC 3'

GATGCCAGATGTGAGATCGTTCTCA

CGCAGTCT 3' 5' GCGGGATCCGAATTCTCTAGAATTA*

ACACTCTCCCCTGTTGAAGCTCTTT

GTGACGGGCGAACTCAG 3' 5' GCGCgA-ATTCACCATGGGTGTGCCC ACT CAGGTCCTGGGGTTGCTGCTGC 31 5' .AGACTGCGTGAGAACGATCTCACAT CTGGCATC 3' 116 (151)6 LC-5 5' GCGCAAGCTTACCATGGGTGTGCCC

ACTCAGGTCCTGGGGTTGCTGCTGC

10 a F Forward Primer B Backward Primer the Sst I cloning site is single underlined 2 the primers, HC-2 and HC-4 contain complementary sequences 3 the EcoR I cloning site is single underlined 4 in HC-4, the G that is double underlined was altered from an A to eliminate a Bgl II restriction site; in HC-5, the C that is doubleunderlined was altered from a T to eliminate a Bgl II restriction site 5 the base A that is double underlined was introduced in the PCR primer to alter the encoded amino acid from an arginine, R, to a serine, S 6 the HindIII cloning site is single underlined 3) Insertion of Modified b12 Heavy Chain Variable Region into the Mammalian Expression Vector The modified b12 heavy chain variable region PCR product was ligated into a mammalian expression vector (Adair, et al.,.Hum.

Antibod. Hybridomas, in press). The mammalian expression vector consisted of the pSG-5 vector (Figure 24) with a human IgGI gene inserted at the EcoR I site. The human IgGI gene contained a VH insert in the same reading frame as the constant regions of the human IgGI gene. The VH insert was removed by digestion with EcoR I and Sst I enzymes.

The constant regions (CH1, CH2, and CH3) remained in the pSG-5 vector. Transcription of the heavy

I

~~=.rtVrTh-t.v~..c 117 chain gene in the pSG-5 expression vector is under the control of the SV40 early promoter.

Transcriptional termination is signaled by the polyadenylation signal sequence downstream of the heavy chain sequence. The M13 intergenic region allows for the production of single-stranded

DNA

for nucleotide sequence determination.

The modified b12 heavy chain variable region PCR product was digested with EcoR I and Sst I and purified on a 2.5% Nu-Sieve agarose gel (FMC). The mammalian expression vector DNA containing the IgGl sequences was digested in parallel with EcoR I and Sst I enzymes to remove the original VH region.

The PCR modified heavy chain variable region was ligated to the constant regions in the mammalian expression vector using T4 DNA ligase under conditions well known to those of skill in the art and transformed into DH5a competent cells following the manufacturer's recommended procedures (GIBCO, BRL Life Technologies, Gaithersburg, MD). The PCR modified heavy chain variable region was inserted in the same reading frame as the constant regions :'of the human IgG1 gene in the pSG-5 vector.

Miniprep DNAs were analyzed and large scale plasmid preparations performed. The nucleotide sequence of the 5' untranslated region including the Kozak sequence, mouse B72.3 heavy chain leader sequence, heavy chain variable region, heavy chain constant regions, and SV40 signal sequence was determined by the dideoxy-nucleotide chain termination method (Sanger et al., suDra) b. Construction of a b12 Liaht Chain Mammalian Expression Vector 1) Modification of b12 Light Chain to Introduce a Kozak SeQuence, Mammalian Leader Sequence, and Human Light Chain Consensus Seauence r 118 The b12 light chain was cloned into a separate pSG-5 expression vector (Green et al., supra) The double-stranded Fab b12 DNA was used as a template for isolating the gene encoding the light chain of the Fab b12, the amino acid residue sequence the light chain of Fab b12 is listed in SEQ ID NO 97. Mouse .B73.2 IgGl DNA (Whittle, et al., Protein Eng., 1:499 (1987) and Bruggmeman, et al., J. Exp. Med., 166:1351 (1987)) was used as a 0 template for isolating the mouse B73.2 leader sequence. Fab b12 and mouse B73.2 IgG1 DNA were thus used as templates for a PCR amplification for the construction of a DNA fragment consisting of the unique Kozak sequence for control of light 15 chain expression, the mouse B72.3 light chain leader sequence (MGVPTQLGLLLWLTDARC (SEQ ID NO 153 from amino acid residue sequence 1 to and the b12 light chain beginning with a human light chain amino acid consensus sequence. (EIVLTQSP (SEQ ID NO 153 from amino-acid residue sequence 21 to 28)).

Altering the beginning of the light chain from the mouse amino acid consensus sequence to the human •amino acid consensus sequence also destroys the original Sac I cloning site. The restriction site, EcoR I, was introduced in the amplification reactions and was located at both the 5' and 3' ends of the fragment. The procedure for creating this fragment by combining the products of two separate PCR amplifications is described below.

The primer pair, LC-l (SEQ ID NO 163) and LC-2 (SEQ ID NO 164), was used in the first PCR reaction as performed above to amplify the Fab b12 light* chain gene and incorporate the human light chain consensus sequence into the fragment .and the EcoR I cloning site into the 3' end of the b12 light chain fragment. For the PCR reaction, 1 gl containing 100 ng of Fab b12 DNA was admixed with 10 41 of loX PCR buffer in a 0.5 ml microfuge tube. To the DNA vs> ~s V 119 admixture, 8 1p of a 2.5 mM solution of dNTPs (dATP, dCTP, dGTP, dTTP) was admixed to result in a final concentration of 200 gM of each dNTP. 1 (equivalent to 20 pM) of the LC-1 primer and 1 pl (20 pM) of the 3' backward LC-2 primer was admixed into the DNA solution. To the admixture, 73 pl of -sterile water and 2.5 units of Taq DNA polymerase was added. Two drops of mineral oil were placed on top of the admixture and 35 rounds of PCR 10 amplification in a thermocycler were performed.

The amplification cycle consisted of 52 0 C for 1 minute, 72 0 C for 2 minutes and 94 0 C for minutes.

The primer pair, LC-3 (SEQ ID NO 165) and LC-4 (SEQ ID NO 166) as shown in Table 10, was used in a separate PCR reaction to amplify the mouse light chain B72.3 leader sequence and incorporate an EcoR I cloning site at the 5' end of the fragment and to introduce a' 27 base pair sequence which has 20 homology to the modified light chain fragment prepared above. Double-stranded DNA encoding the

U

mouse B73.2 IgGl (Whittle, et al., supra) was used as a template for preparation of the mouse 72.3 leader sequence. The PCR reaction to prepare the i mouse leader sequence fragment was performed using the same conditions as described in Example 4a .for the preparation of the modified VH fragment.

The resultant PCR modified b12 light chain DNA fragment and light chain mouse leader sequence fragment were purified by electrophoresis in a Nu-Sieve agarose gel (FMC). The area in the agarose containing the modified b12 light chain DNA fragment and light chain mouse leader sequence fragment.were excised from the agarose.

A third PCR amplification using the primer pairs, LC-1 (SEQ ID NO 157) and LC-4 (SEQ ID NO 166) as shown in Table 10, was performed to fuse the light chain mouse leader fragment with the 120 modified light chain fragment. The primers used for this. amplification were designed to preserve an EcoR I site, a unique Kozak sequence, and the mouse B72.3 light chain leader sequence on the 5' end of -the amplified fragment and to preserve the EcoR I cloning site on the 5' end of the amplified fragment. The templates used in this PCR reaction were the two purified PCR reaction products described above. The PCR reaction and subsequent purification of the PCR product were performed as described in Example 4al.

2) Insertion of Modified b12 Light Chain into pSG-5 Mammalian Expression Vector The modified b12 light chain PCR product was ligated to a pSG-5 vector (Figure 24).

The pSG-5 vector had the same features described in Example 4a2 but did not contain a human IgGl gene.

The modified b12 light chain PCR product was digested with EcoR I and purified on a 2.5% Nu- Sieve agarose gel (FMC). The pSG-5 vector DNA was digested in parallel with EcoR I enzyme. The PCR modified light chain was ligated to the vector using T4 DNA ligase (New England Biolabs, Beverly, MA) and transformed into DH5a competent cells (GIBCO, BRL Life Technologies, Gaithersburg, MD) following manufacturer's instructions.

Miniprep DNAs' were analyzed and isolation of plasmid DNA performed. The nucleotide sequence of the light chain gene was determined using the dideoxy-nucleotide chain termination method (Sanger et al., supra). The nucleotide sequence of the untranslated region, mouse B72.3 light chain leader sequence, light, chain variable region, light chain constant region, and SV40 signal sequence was obtained. The nucleotide and amino acid residue sequences are illustrated in Figures 25A and 121 and are given in the sequence listing as SEQ ID NOs 152 and 153.

c. Transient Expression of b12 Heavy and Light Chain Genes in pSG-5 Vectors in COS-7 Cells 1) Transient Expression of b12 IqG1 in COS-7 Cells The human heavy and light chains in *10 the separate pSG-5 expression vectors were cotransformed and transiently expressed in COS-7 cells. COS-7 cells (SV40 transformed African Green Monkey Kidney Cells) provide a rapid and convenient method to test the expression'and function of the antibody genes. The COS-7 cells constituitively i:! express the SV40 large T antigen which supports the transient replication of episomes carrying the origin of replication. The pSG-5 expression vector has an SV40 origin of replication. Upon 20 transfection into COS-7 cells, the expression vectors are.replicated in the nucleus to a high copy number, resulting in relatively high transient i expression levels.

COS-7 cells were obtained from the American Type Culture Collection (CRL 1651) and cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (GIBCO BRL, Gaithersburg, MD) and 1% penicillin, and 1% streptomycin. Transfections were performed with Ag of plasmid DNA per 100 mm tissue culture plate containing 1 x 106 cells. The control plate was transfected with plasmid vector DNA without an insert. The plates were incubated at 37 0 C after transfection. The supernatants were harvested at 48 hours and tested for gpl20 binding specificity in an ELISA assay.

.Xa 122 2) ELISA Assay for the Detection of Binding of b12 IqG1 to Supernatants from COS-7 transformants were tested for binding to gpl20 in an ELISA assay. Briefly, the ELISA plate was .coated with recombinant IIIB gpl20 antigen at a concentration of 1 Ag/ml. The serially diluted supernatant containing the b12 antibody was added to the wells and incubated at 37 0 C for 1 hour.

10 After washing the plate to remove unbound antibody, a goat anti-human Ig Fc horse radish peroxidase (HRP) conjugated secondary antibody was added and incubated for an additional hour. An OPD substrate for the HRP conjugated antibody was added and the 15 HRP activity detected by determining the absorbance at 490 nm.

d. Insertion of the b12 Heavy Chain IqG1 'into the pEE6 Mammalian Expression Vector to Create pEe6HC BM 12 After confirmation that the antibody molecule expressed by the heavy and light chain expression vectors bound gpl20 as described in Example 4c, the heavy chain was.removed from the pSG-5 vector and ligated into the pEE6 mammalian expression vector (Bebbington et al., Bio/Technoloqy, 10:169 (1992)). The pEE6 vector (Celltech, England) contains an HCMV promoter and the glutamine synthetase gene The pEE6 vector was chosen because of the GS gene which serves as a selectable marker. CHO cells are devoid of GS activity and thus are dependent on a supply of glutamine in the culture medium. Cells transfected with the pEE6 vector containing the GS gene are able to synthesize glutamine from glutamate and can survive in the absence of glutamine in the culture medium. For CHO cells, the addition of methyl sulfoxamine (MSX) leads to 123 amplification of the transfected plasmid

DNA.

The heavy chain pSG-5 vector was digested with EcoR I and Bgl II to remove the 5' untranslated region including the unique Kozak sequence, mouse heavy chain B72.3 leader sequence, and heavy chain variable and constant regions from the vector. The pEE6 vector was also digested with EcoR I and BamH I. Both the vector and heavy chain DNAs were analyzed on a 0.7% low melting point 10 agarose (LMPA) gel. The 3.5 kb heavy chain band and the 4.68 kb pEE6 vector band were excised from the gel and ligated together in the presence of the LMPA at 15 0 C overnight with 1 il of T4 DNA ligase -and 1 Al of 10X ligase buffer (New England Biolabs, Beverly, MA). Upon ligation, the EcoR I site is S0:0. reconstituted but the BamH I and BglII sites are destroyed. Prior to transformation, 5 P1 of the ligated DNA in LMPA was diluted with 20 pi of TCM buffer (10 mM tris, 10 mM CaCl 2 and 10 mM MgC1) 20 Only 10 Il of the 25 1l was used for the transformation. The ligated circular plasmid DNA construct was transformed into maximum efficiency DH5a competent cells. The standard protocol for transformation was used, wherein the DNA and 100 l :i of the competent bacterial mix (GIBCO BRL, Gaithersburg, MA) were incubated on ice for minutes and heat shocked at 420C followed by incubation on ice for 2 minutes. About 900 pl of SOC (GIBCO BRL, Gaithersburg, MA) was added to the transformation. Only 100 pl of the 1000 il of the transformed cells was plated on LB with carbenicillin plates (carbenicillin at 50 Ag/ml).

The plates were incubated at 37 0 C overnight.

Twelve individual colonies were picked for miniprep analysis. Several diagnostic digests confirmed the presence of the heavy chain insert. Plasmid DNA was isolated on a CsCl gradient (Sambrook et al., supra). The nucleotide and amino acid residue 124 15 0 3O* sequences are illustrated in Figures 27A through 27E and the nucleotide and amino acid residue sequences are given in the sequence listing as SEQ ID NOs 154 and 155.

e. Insertion of the b12 Light Chain into the pEE12 Mammalian Expression Vector The light chain was ligated into the pEE12 vector (Celltech, England) from the vector involving similar steps as described in Example 4d for the heavy chain. The pEE12 vector has a human CMV promoter for expression of the light chain, a polylinker to provide cloning sites, and a polyadenylation signal for termination of transcription. The vector also contains the GS selectable marker gene, whose expression is controlled by an SV40 early promoter at the 5' end of the GS gene, an intron, and a polyadenylation signal at the 3' end of the GS gene.

1) Preparation of Modified b12 Light Chain The 5' PCR primer was designed to replace the EcoR I cloning site with a HindIII cloning site. The 3' PCR primer maintained the EcoR I cloning site.

The primer pair, LC-5 (SEQ ID NO 167) and LC-2 (SEQ ID NO 165), was used in the PCR reaction as described in Example 4al to amplify the Fab b12 light chain gene and incorporate HindIII and EcoR I cloning sites into 5' and 3' ends of the fragment, respectively. The b12 pSG-5 vector containing the b12 light chain was used as the template in the PCR reaction. For the PCR reaction, 1 Al containing 100 ng of b12 pSG-5 DNA was admixed with 10 Al of PCR buffer in a 0.5 ml microfuge tube. To the DNA admixture, 8 il of a 2.5 mM solution of dNTPs (dATP, dCTP, dGTP, dTTP) was admixed to result in a 125 final concentration of 200 micromolar (AM) of each dNTP. 1 il (equivalent to 20pM) of the LC-5 primer and 1pl (20 pM) of the 3' backward LC-2 primer was admixed into the DNA solution. To the admixture, 73 pl of sterile water and 2.5 units of Taq DNA -polymerase was added. Two drops of mineral oil were placed on top of the admixture and 35 rounds of PCR amplification in a thermocycler were performed. The amplification cycle consisted of 52 0 C for 1 minute, 720C for 2 minutes and 940C for 0.5 minutes.

The resultant PCR modified b12 light chain DNA fragment was purified by electrophoresis in a Nu-Sieve agarose gel (FMC). The area in the 15 agarose containing the modified b12 light chain DNA fragment was isolated from the agarose.

2) Insertion of the Modified b12 Light Chain into the DEE12 Mammalian 20 Expression Vector The modified b12 light chain i purified PCR product and the pEE12 vector were digested.with HindIII and EcoR I in separate reactions.. The digested DNAs were analyzed on an LMPA gel, the DNA excised, and ligated together in the presence of the LMPA gel as described for the heavy chain construct in Example 4d. The ligation.

products were transformed into DH5a competent cells, minipreps analyzed, and DNA prepared as described for the heavy chain constructs in Example 4d.

f. Insertion of the Modified b12 Heavy Chain into the pEE12 Mammalian Expression Vector Containing the b12 Light Chain to Create the Combinatorial Vector DEel2 Combo BM 12 A heavy chain cassette comprising the

-I-

126 HCMV promoter, enhancer elements, heavy chain gene, and polyadenylation signal were removed from the pEE6 vector and inserted into the pEE12 vector containing the b12 light chain gene, prepared in Example 4e, to generate the combinatorial construct, pEel2 Combo BM 12, containing both the b12 light and heavy chain genes (Figure 28).

The heavy chain cassette was removed from the pEE6 vector by digestion with BglII and Sal The pEE12 vector containing the light chain gene, prepared in Example 4e, was also digested with BglII and Sal I. The heavy chain cassette and the *^-pEEl2 vector containing the light chain gene from Example 4e were ligated together at the BglII and Sal I sites as described in Example 4d. The combinatorial construct was transformed into competent cells and miniprep DNA was analyzed for the presence of the heavy and light chains as in Example 4d. The nucleotide sequence of the heavy and light chain genes was determined. The nucleotide sequence of pEel2 Combo BM 12, the pEE12 vector containing the b12 heavy and light chain genes is given in the sequence listing as SEQ ID NO S"156 and is illustrated in Figures 29A through 29R.

g. gpl20 Binding of b12 IaG1 Antibody Exoressed from the Heavy and Light Chain Genes in the Combinatorial Vector pEel2 Combo BM 12 The combinatorial pEel2 Combo BM 12 vector containing both the heavy and light chain genes was used to transfect CHO cells. Stable clones were selected in Glasgow Minimal Essential Media (GIBCO) supplemented with 10% dialyzed fetal bovine serum and 50 pM methyl sulfoxamine (MSX).

Several clones were isolated and expanded in 6-well cluster dishes. The supernatants of subconfluent cultures were harvested and tested by ELISA for 127 binding to gpl20 as described in Example 4c2. The clone producing the highest levels of b12 IgGl as determined by ELISA with gpl20 IIIB was chosen for further study. The antibody was purified by affinity chromatography using protein A as described in Sambrook, et al., supra. The affinity of b12 IgGl for gpl20 IIIB as measured by surface plasmon resonance as described in Example 2b6c is 1.3 x 109 M Neutralizing Activity of Recombinant b12 Whole IqGl Antibody (b12 IqG!) Aaainst HIV-I In Vitro The key issue in producing antibodies to HIV-l for therapeutic or prophylactic purposes is that they should be highly potent (of high affinity and neutralizing ability) and be cross reactive with a wide range of primary clinical (field) isolates.

These are generally two opposing characteristics.

The ability of b12 whole IgG1 antibody (b12 IgGl) to neutralize the infectivity of laboratory strains of HIV-l and a wide variety of primary clinical isolates has been examined in p24 ELISA assays, -microplaque assays, and by syncytial formation assays.

The primary clinical isolates used as a source of HIV-l virus in these assays came from various regions of the world by three organizations: the World Health Organization (WHO), the Henry M.

Jackson Foundation for the Advancement of Military Medicine (HMJFAMM), and the National Institute of Allergy and Infectious Diseases (NIAID). Isolates from the WHO Network for HIV-I Isolation and Characterization were obtained through the AIDS Research and Reference reagent Program, Division of AIDS, NIAID, NIH. Isolates from HMJFAMM were provided by Dr. John Mascola, Walter Reed Army Institute of Research, Rockville, MD and Dr.

128 Francine McCutchan, Henry M. Jackson Research Laboratory, Rockville MD. Isolates from NIAID were kindly provided by Dr. Jim Bradac, Division of AIDS, NIAID, NIH.

The HIV-1 viruses were collected from various '-regions of the world, expanded in mitogenstimulated peripheral blood mononuclear cells (PBMC) (Mascola et al., J. Infect. Dis., 169:48-54 (1994)), and culture supernatants containing \'20 infectious virus were stored in central repositories at -70°C. The designation of viruses into clades was made on the basis of sequence information based on the gaq gene or on the region of gpl20, or in some cases, after heteroduplex mobility analysis (Louwagie et al., AIDS, 7:769-772 (1993) and Delwart et al., Science, 262:1257-1261 (1993)).

The HIV-1 viruses include a set of 14 primary isolates which contain a high proportion of 0 isolates.which are relatively refractory to antibody neutralization by sera from other HIV-l S infected individuals (Wrin et al., J. Aca. Imm. Def. Synd., 7:211-219 (1994)), 12 primary infant "isolates obtained at birth or within two weeks of age, and 69 international isolates belonging to 6 different clades.

Several different neutralization assays were performed because HIV-1 neutralization by antibody shows considerable variation depending upon the assay used and the precise experimental conditions such as inoculum size and incubation time of virus and antibody (D'Souza et al., AIDS, 8:169-173 (1994)). By performing neutralization assays on a range of laboratory and primary isolates in a number of different. laboratories, it has been demonstrated that b12 IgGi is a highly potent neutralizing antibody effective against a wide breadth of isolates.

129 a. Quantitative Neutralization of HIV-1 MN and IIIb by b12 IgGl as Measured in a Plaque Assay b12 IgG1 was initially tested for its ability to neutralize the HIV-1 laboratory strains MN and IIIB in a plaque formation assay in laboratories which recently tested a panel of monoclonal antibodies as part of the NIAID/WHO Antibody Serological Project (D'Souza et al., supra).

b12 IgG1 showed 50% neutralization titers of 3 ng/ml for the MN strain and 7 ng/ml for the IIIB strain using plaque formation (Hanson, et al., J.

Clin. Microbiol., 28:2030-2034 (1990)) to determine the ability of the antibody to inhibit infectivity of the HIV-1 strains.

b. Quantitative Neutralization of HIV-1 MN and IIIb by b12 IqG1 as Measured by Syncytial Formation o ''b12 IgG1 showed 50% neutralization titers.,: of 20 ng/ml for both MN and IIIB strains using syncytial formation as the reporter assay as described in Example 3b (Nara et al., AIDS Res.

25 Human Retroviruses, 3:283-302 (1987)).

The syncytial formation assay was performed as described.in Example 5c. Briefly, virus was grown in H9 cells. For infectivity measurement, monolayers of CEM-SS target cells were cultured with 100-200 syncytial forming units (SFUs) of virus, in the presence or absence of antibody, and the number of syncytia determined after 3-5 days of incubation. The assays were repeatable over a virus-surviving fraction range of 1 to 0.001 within a 2 to 4-fold difference in the concentration of antibody (P<0.001).

I- l~il-r 130 c. Neutralization of Primary Virus Isolates by b12 IqGl as Measured by the p24 ELISA Assay The ability of b12 IgGI to neutralize ,.infectivity of PBMCs by HIV-1 virus was -quantitatively measured in the p24 ELISA assay (Daar et al., Proc. Natl. Acad. Sci. U.S.A., 87:6574-6578 (1990) and Ho et al., J. Virol., 65:489-493 (1991)). The p24 ELISA assay is further 0 described in Example 3a.

Neutralization of Ten Primary Virus Isolates by b12 IqGl HIV-1 viruses were isolated from individuals from various locations in the U.S. and with varying disease status. The HIV-1 viruses had "been cultured only once or twice in peripheral blood mononuclear cells (PBMCs). Viral stocks were grown in PBMCs and the assay was performed in PBMCs.

Briefly, HIV-1 virus at 50 TCID 5 0 and varying concentrations of b12 IgGl were incubated together for 30 min at 37°C before addition to PHAstimulated PBMCs. HIV-1 virus replication was assessed after incubation for 5 to 7 days by p24 ELISA measurement as described in Example 3a. HIV- 1 virus positive controls used in this assay were the molecularly cloned HIV-1 virus JR-CSF and the HIV-1 isolate-JR-FL (O'Brien et al., J. Virol., 66:3125-3130 (1992), O'Brien et al., Nature, 348:69-73 (1990), and O'Brien et al., J. Virol., in press (1994)). Stocks of JR-CSF were prepared by infection of PBMC with supernatants initially obtained by DNA transfection. -HIV-1 IIIB and HIV-1 MN are viruses with an extensive history of passage in transformed T-cell lines (Robert-Guroff et al., Nature, 316:72-74 (1985)). Stocks of these strains grown in H9 cells were passaged in mitogen- 131 stimulated PBMC to prepare viruses that had been grown in the same cells as the primary viruses, to eliminate the influence of any host cell-dependent epigenic factors on virus neutralization (Wrin, et al., J. Aco. Imm. Def. Synd., 7:211-219 (1994)).

The stock of PBMC-grown MN was a gift from A. N.

-Conley (Merck Research Labs).

2) Neutralization of 12 Primary Infant Isolates by b12 IqGl b12 IgGi was also tested for the ability to neutralize infectivity of a panel of 12 primary infant isolates in the p24 ELISA assay.

Virus isolates were obtained from 12 infants born to HIV-1 seropositive mothers; 7 were obtained at birth and 5 between birth and 14 days of age. All the infants were from California. Virus was S" isolated from patient PBMCs by coculture with PBMCs from healthy seronegative donors. Viral stocks were prepared by passaging the last positive culture dilution once into PBMCs. All of the isolates, except one (isolate were nonsyncytial inducing in MT2 cells and therefore could not be assayed in the syncytial forming assay as herein described. HIV-1 virus from these stocks was grown in PBMCs and neutralization assessed using PHA-stimulated PBMCs as indicator cells and determination of extracellular p24 as the reporter assay.essentially as described in Example 3a (AIDS Clinical Trials Group Virology manual for HIV Laboratories, Department of AIDS Research,

NIAID,

NIH, version 2.0 (1993)).

Serial dilutions of b12 IgGI (0.3 to 20 Ag/ml) were incubated with 20 TCID 5 0 or 100 TCID 5 0 virus in 35 triplicate for 2 hours at 37 0 C before addition to PHA-stimulated PBMCs. Virus replication was assessed after 5 days by p24 ELISA measurement.

Neutralization was expressed as either a 50% or 132 reduction in p24 antigen as compared to values observed in the absence of antibody (Table 6).

d. Neutralization of Primary Virus Isolates by b12 IqGl as Measured in a Microplaque Assay A quantitative microplaque assay to measure the reduction of infectivity of primary clinical isolates of HIV-1 in the presence of the ."10 b12 IgGl and pooled human plasma was performed as described in Hanson et al., J. of Clin. Microb., 2030-2034 (1990). The set of primary clinical isolates was chosen to contain a high proportion of isolates which are relatively refractory to antibody neutralization by sera from other HIV-1 infected individuals (Wrin et al., J. Aca. Imm.

Def. Svnd., 7:211-219 (1994)). Viruses were grown in PBMCs and the assay carried out in MT2 cells.

S* This limits study to viruses which grow in this cell line but provides an additional measure neutralization.

Primary clinical isolates of HIV-1 were isolated from frozen peripheral blood lymphocytes obtained from seropositive donors as described in Gallo et al., J. of Clin. Microb., 1291-1294 (1987) and cultivated in peripheral blood mononuclear cells (PBMC). Briefly, HIV isolates were obtained by incubating frozen HIV-infected patient PBMCs with seronegative donor PBMCs in RPMI-1640 medium containing 20% heat-inactivated fetal bovine serum, 2 ig/ml polybrene, 5% interleukin-2, and 0.1% antihuman leukocyte interferon. The cultures were fed with fresh donor PBMCs once a week, and the supernatants were assayed for the presence of reverse transcriptase (RT) activity beginning at day 11. The cultures were considered positive if, for 2 consecutive weeks, the RT counts were fold higher than those in the cultures of the 133 seronegative donor PBMCs alone.

The resultant RT-positive virus isolates were tested for cytolysis in the MT4 (U-4 clone) (Hanson et al., supra). Cytolysis in MT4 is a requirement for viruses to be usable in the subsequent MT2 microplaque assay system. Supernatant fluids from -the primary PBMC isolation cultures were used to infect expanded cultures of phytohemagglutinin (PHA)-stimulated PBMCs from healthy seronegative blood donors. These infected PBMC cultures were grown in RPMI-1640 medium supplemented with fetal bovine serum, 5% interleukin-2, 0.1% anti-a interferon, 2 Ag/ml polybrene, 50 Ag/ml gentamicin, 100 U/ml penicillin, and 100 pg/ml streptomycin.

15 The crude supernatants were harvested after 7 days and.frozen as viral stocks at -70 0

C.

The primary clinical isolates of HIV-1 used in this microplaque assay are given in Table 6.

VL134, VL648, and VL025 are viruses isolated from 20 infected mothers in New York in 1992; UG266 and S/ UG274 are clade D isolates which were a gift from John Mascola the Division of Retrovirology, Walter Reed Army Institute of Research; the remaining viruses were isolated from homosexual males in California in 1992. The pooled human plasma preparation, containing neutralizing antibody, was derived from 13 HIV-1 positive individuals selected for high neutralization titer against the MN isolate. The laboratory HIV-1 strains MN and IIIb were propagated in H9 cells as controls in the microplaque assay.

b12 IgGl and a pool of human plasma from 13 HIV-1 seropositive patients were used as the source of neutralizing antibodies in a 96-well microtiter plaque reduction assay as described by Hanson et al., supra. Briefly, 3-fold serial dilutions of the b12 IgG1 or heat-inactivated pooled patients' plasma were combined in quadruplicate with an equal t-44.4-A. 134 volume containing 20 plaque-forming units (PFU) of HIV-I virus per well and incubated for 18 hours at 37 0 C. Negative control wells also. contained normal human serum pool with no patient immune.

serum. After the 18 hour incubation of Fabs or serum and virus, 90,000 MT2 cells were added per well and incubated at 37 0 C for 1 hour. SeaPlaque Agarose in assay medium at 39.5C was then added to a final concentration of While the warm agarose was still molten, the microtiter plates were centrifuged at 20 0 C for 20 minutes at 500 X g form cell monolayers. The plates were incubated for 6 days at 37 0 C and then stained 18 to 24 hours with 50 pg/ml propidium iodide. The fluorescent :15 plaques were counted with transillumination by a 304 nm ultraviolet light source using a low-power stereo zoom microscope. Inhibition of infectivity, or neutralization titer, is defined as the pg/ml of Fab or the plasma dilution giving 50% inhibition of plaque count as compared with controls without antibody. This dilution was interpolated between data points.

e. Results of the Neutralization Assays by b12 IqGl with Laboratory Virus Isolates Results of the ability of the b12 IgG to neutralize laboratory virus isolates in both the plaque and syncytial formation assays suggest the antibody is approximately two orders of magnitude more potent than other CD4 site antibodies in the WHO/NIAID Project and comparable to the best antibodies directed to the V3 loop of gpl2O.

However, whereas antibodies directed to the V3 loop of gpl20 are strongly strain specific, b12 IgGl is roughly equally effective against MN and IIIB. The b12 IgGl antibody is comparable in potency to a CD4-IgG molecule in these assays (Example 3c). In a separate assay using p24 production to determine S~ 135 infectivity (Daar et al., Proc. Nati. Acad. Sci.

87:6574-6580 (1990) and Ho et al., J.

Virol., 65:489-493 (1991)), 50% neutralization titers of less than 40 ng/ml were found for both the MN and IIIB laboratory strains.

f. Results of the Neutralization Assays by b12 IqGl with Primary Virus Isolates b12 IgGI showed essentially complete i0 neutralization of 7 of 10 isolates at 5 Ag/ml with ;all the isolates showing 50% neutralization at f1 ig/ml as determined in the p24 reporter assay (Figure 21).

The inhibition of infectivity, or neutralization titer, for b12 IgGl and the pooled 1'.HIV seropositive human plasma from 13 donors is given in Table 6. The neutralization titer for each of the viral isolates is expressed as the minimum pg/ml of b12 IgGl required for 50% 20 inhibition of plaque count as compared to the controls. The neutralization titer for each of the ;...viral isolates is expressed as the minimum titer of the pooled HIV seropositive human plasma from 13 donors required for 50% inhibition of plaque count as compared to the controls.

136 Table 6 virus

IIIB

MN

15 15 VL135 UG274 VL134 VL596 UG266 VL434 VL172 VL750 VL069 VL077 VL114 VL263 VL648 VL025 host cell H9 H9

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

PBMC

b12 IgG1 50% neutralization titer (ug/ml) 0.007 0.003 10 0.7 5.6 8.5 3.8 22 >200 >200 >50 >200 <7.4 5.0 16.7 16.7 1:44 1:37 1:30 1:17 1:12 1:10 1:10 1:10 <1:10 <1:10 <1:10 <1:10 <1:10 <1:10 pooled human plasma: dilution for neutralization 1:767 1:24,000 i i The b12 IgG1 was able to neutralize ten of the fourteen primary clinical isolates assayed at concentrations of s50 pg/ml as measured as the yg/ml required for 50% inhibition of plaque count as compared to the controls (Table Pooled human plasma was able to neutralize 5 of the 14 primary ias s s a l.

137 clinical isolates assayed at >1:10 dilution as measured as the dilution required f or 50% inhibition of plaque count as compared to the controls without antibody.

Table 6 shows that four isolates, which were not neutralized even by a 1:10 dilution of-pooled human plasma, were neutralized by b12 IgGl. Most of the viruses reported in Table 6 were isolated from U. S.

donors although two, both of which are neutralized by b12 IgGl, were from Ugandan donors and assigned to dade D.

Results of neutralization of 12 infant primary isolates with b12 IgG1 as determined by p24 ELISA measurements are given in Table 7.

Table 7 .b12 IgGl Antibody Concentration ig/ml) Infant Isolate 50% inhibition >90% inhibition 1 20 2. 1.25 3 <0.3 0.3 4 <0.3 0.6 2.5 6 5 7 5 8 <0.3 0.3 9 0.3 0.3 11 <0.3 0.6 12 .0.3 138 As shown in Table 7, bl2 IgGi achieved neutralization for 8 of 12 infant isolates at concentrations of s20 Ag/ml in the p24-based assay.

All 12 isolates were 50% neutralized in the range of 0.3 to 20 g/ml with the majority being Sneutralized at <5 pg/ml. In contrast, a pooled hyperimmune globulin product HIVIG achieved neutralization of only 3 or 12 isolates within a 4 concentration range up to 100 Ag/ml. HIVIG is a 10 hyperimmune IgG preparation obtained from the :'"'"pooled plasma of selected HIV-1 asymptomatic seropositive donors meeting the following criteria: presence of p24 serum antibody titers >128, CD4 lymphocyte count a400 cells/Al and the absence of :'15 p24 and hepatitis B surface antigen by enzyme immunoassay (Cummins et al., Blood, 77:1111-1114 (1991)). The HIVIG used in these experiments was lot number IHV-50-101 (North American Biologicals).

4. 4 HIV-1 neutralization by antibody shows considerable variation depending upon the assay used and precise experimental conditions such as inoculum size and incubation time of virus and antibody (D'Souza et al., supra). However, by carrying out neutralization on a range of laboratory and primary isolates in a number of assays in different laboratories, we have shown that b12 IgGl is a highly potent neutralizing antibody effective against a wide breadth of primary isolates. The results clearly demonstrate that, although primary isolates may be more difficult to neutralize by antibody than laboratory strains, they are not intrinsically resistant (Conley et al., Proc. Nati. Acad. Sci.. U.S.A., 91:3348-3353 (1994)). The potency of b12 IgGl against the majority of U. S. isolates is in a concentration range (55 Ag/ml) which could be achieved in vivo in passive immunotherapy.

Furthermore, the affinities of recombinant 139 antibodies displayed on phage can be enhanced by mutagenesis and selection in vitro and this strategy has been used to considerably improve the potency and breadth of reactivity of Fab b12 (Barbas et al., Proc. Natl. Acad. Sci., U.S.A., S91:3809-3812 (1994)). For optimal potency and strain cross-reactivity for passive immunization, a cocktail of in vitro improved antibodies may be most appropriate.

10 The results have implications for passive immunization and vaccine design. The ability of b12 IgG1 to neutralize a range of primary isolates implies conservation of a structural feature associated with the CD4 binding site of gpl20 which is accessible to antibody and important for neutralization. A vaccine might seek to present this feature to the immune system. Clearly, the feature is present on recombinant gpl20 since b12 was affinity selected from a library using.this 20 .molecule.. However, b12 and related antibodies formed only a small part of the repertoire affinity selected from this library by recombinant SMost of the antibodies obtained were far less potent in neutralization even though they were also directed to the CD4 binding site, were crosscompetitive with b12 for binding to recombinant gp120 and had similar affinities to bi2 (Barbas et al., Proc. Natl. Acad. Sci.. 89:9339-9343 (1992), Barbas et al., J. Mol. Biol., 230:812-823 (1993), and Example 2b6) Therefore, recombinant gpl20 appears to present the b12 epitope in conjunction with several other weakly neutralizing and overlapping epitopes and its efficacy as a vaccine may suffer. Interestingly, evidence from antibody binding to infected cells suggests that b12 does recognize a native conformation of gpl20 more effectively than other CD4 binding site antibodies (Example In any 1 140 case, b12 IgG1 and the library approach could.be useful in vaccine and passive immunization evaluation. The ability of a candidate vaccine to preferentially bind b12 and/or preferentially select potent neutralizing antibodies from libraries should be positive indicators for vaccine development.

5. Determination of the Relationship Between the :10 Epitopes Recognized by Fabs with Purified HIV- 1 Antigens The Fabs show a spectrum of neutralizing abilities as described in Example 5. It was therefore sought to determine if the epitopes recognized by individual Fabs could be distinguished from each other, and if possible, determine how the epitopes recognized by the individual Fabs related to neutralization.

a. Competitive ELISA between Fabs and b13 Whole IqGl Antibody for Binding to The first method to distinguish between the epitopes bound by the Fabs of this invention was to compare the epitope recognized by the Fab b13 with the other Fabs. The Fab b13 had been spliced to. the Fc region of IgG1 to generate a whole IgGl molecule and therefore contains the Fc region of the IgG1 antibody. The other Fabs do not contain the Fc region of the IgGi antibody. The binding of the b13 IgGl could therefore be distinguished from the binding of other Fabs by using a labeled anti-Fc reagent in competition ELISA. A competition ELISA in which the Fabs b3, b6, bll, b12, and b14 competed with b13 IgG1 for binding to immobilized gpl20 was performed.

Competitive ELISAs were performed between the Fabs b3, b6, bll, b12, and b14 and the b13 whole IgG1 antibody. The whole antibody was obtained by 1 141 splicing constant domain genes with the b13 Fab and expressing the protein in Chinese Hamster Ovary cells (CHO) as described in Example 4g (Bender et al., supra and in Example 4a for the Fab b12). The ELISA was performed as described above in Example 2b6)(b). Briefly, microtiter wells were coated -with 0.1 jig/ml of gpl20 derived from the HIV-1 strain LAI in 0.1 M bicarbonate buffer at pH 8.6.

Soluble or free Fab fragments were serially diluted from 1:100 to 1:32,000 in 0.5% BSA/0.025% Tween 20/PBS. The dilution of b13 IgG1 was held constant at 1:10,000 in 0.5% BSA/0.025% Tween 20/PBS. The b13 IgG1 and Fabs were admixed, added to the coated microtiter wells and maintained for 120 minutes at 37 0 C. After maintenance, the wells were carefully washed ten times with 0.05% Tween The amount of b13 IgGi antibody bound to the plate after washing was detected using a peroxidaselabeled antibody specific for the Fc portion of IgG1 contained on the b13 antibody.

Results of this assay indicated that the Fabs b3, b6, bll, b12, and b14 are competitive with b13 IgGl for binding to gpl20 indicating that the epitopes recognized by the individual Fabs are probably either proximal or identical to the epitope recognized by the b13 IgG1. A control anti-tetanus toxoid Fab.did not compete with IgG1 b13 in this assay.

Competition monitored in an ELISA format showed that all of the Fabs compete with the b13 Fab as a whole IgG. There is also an indication that Fabs b12 and b13 are distinct in that they are somewhat less effective in cross-competition than the other members of the panel.

b. EpitoDe Similarity Determination Between the Fabs in Binding to Using BIAcore 142 A more precise method for determining the similarity of epitopes was performed using the BIAcore. The procedure adopted here was to immobilize a polyclonal anti-human F(ab') 2 on the sensor chip and use this to capture -the individual Fabs. An Fab of this invention was injected and captured by the polyclonal anti-human F(ab') 2 The captured Fab was then used to bind gpl20 derived from the HIV-1 strain LAI. The i.:'t0 captured Fab would thus bind the gpl20 at its respective epitope.. A second Fab of this invention was then injected. A response in the BIAcore assay after injection of the second Fab indicates that binding has occurred. If the second Fab injected recognizes -the same or similar epitope on the as the first Fab, no response would occur. No response would therefore indicate that the two Fabs tested in the assay competed for binding to the same or similar epitope on gpl20. Alternatively, a response in the assay suggests that the epitopes recognized by the two Fabs are distinct from one another and that binding of the second Fab to to a second epitope is possible in the presence of the first Fab. A response would therefore indicate that the two Fabs tested in the assay did not compete -for binding to the same or similar epitope.

The precise epitope similarity determination with the BIAcore was performed as follows. A flow rate of 5 pl/min of PBS, pH 7.4 was established and the biosensor chip was activated by injecting 30 il of activation solution (Pharmacia Biosensor, 0.2 M N-ethyl-N'-(e-diethylaminopropyl)carbodiimide, 50% N-hydroxysuccinimide). The flow rate was then adjusted to 10 il/min and the antigen was injected in 10 mM sodium acetate buffer, pH Forty Al of goat anti-human F(ab') 2 (Pierce) at a concentration of 40 Ag/ml in 10 mM sodium acetate buffer, pH 4.5 was injected to give a final 3+VZ-VS4. *N f s 143 immobilization of 10000 Response Units The chip was then blocked from any further immobilization by injecting 30 yl of 1 M ethanolamine, pH 8.5 (Pharmacia Biosensor). The flow rate was adjusted to 1 pil/min and 4 p1 of the *first Fab at a concentration of 100 yg/ml was -injected, immediately followed by 4 il of an anticytomegalovirus Fab at a concentration of 150 /ig/ml to block any remaining binding sites on the immobilized goat anti-human F(ab') 2 Next, 4 i1 of gpl20 at a concentration of 10 ig/ml was injected followed by 4 i of the second Fab at 100 pg/ml.

The assay was performed with a combination of all of the Fabs to give a mosaic of binding patterns.

The entire surface was regenerated with 25 p1 of mnM HCl so. that the next cycle could be performed.

*Table 8b indicates the results of the epitope similarity determination by BIAcore. Table 8a shows the positive and negative controls for the 20 'clones used. The positive controls are the RU levels obtained when the first Fab used is the clone indicated and the second Fab is an anti-gpl20 V3-loop Fab. The Fabs of this invention compete with soluble CD4 for binding to gpl20. The second' Fab, an anti-gpl20 V3-loop Fab, neither competes with soluble CD4 nor competes with anti-CD4 site Fabs and therefore would'react with a different epitope than the Fabs of this invention. As can be seen from the table, all positive controls result in significant values of 125 or more, indicating the validity of the technique to distinguish between non-identical epitopes. The negative controls are the values obtained when the same Fab is injected twice. This gives the background values for each Fab. These values were subtracted from all subsequent experiments in order to give true values.

144 An epitope map, Table 8b, was then constructed. ND indicates that this combination of Fabs was not performed. It can be seen from this map that Fabs b3, b6, bil, and b14 form a set which compete highly effectively with one another for binding to a similar or the same epitope. For the most part, a member-of the set competes for binding as well with another member as it does with itself .(RU On the other hand, b12 and b13 appear somewhat different in that while they compete for binding with members of the above set, they do not compete as effectively as the other Fabs within the set. Further, competition for binding to the same or similar epitope between b12 and b13 is incomplete. This suggests that the epitopes of Fabs b12 and b13 are sufficiently dissimilar from those of the other four and from each other, to :.al'low detectable binding when they are used in combination with any of the other Fabs. It may therefore be concluded that clones b3, b6, bli, and bi4 bind the same or similar epitopes, with Fabs b12 and b13 bind to epitopes which can be distinguished from the other epitopes in this assay.

Table 8a Fab b3 b6 bil b12 b13 b14

POSITIVE

CONTROL (RU) 129 128 131 125 135 134

NEGATIVE

CONTROL (RU) 24 38 ND 17 15 ND ND indicates that this combination of Fabs was not performed.

145 Table 8b Fab 1 b13 b12 b6 b3 b14 30 24 14 0 SFab 2 bll 54 28 14 0 b3 26 29 0 0 Sb6 21 17 0 ND b12 .22 0 ND ND ND indicates that this combination of Fabs was not 10 performed.

c. Comparison of Fab EDitopes with Wild-type and Mutant Forms of qpl20 Using ELISA S* with cpl20 in the Solid Phase Epitope similarity determinations of the panel of Fabs was performed with a panel of HXBc2 gpl20 mutants of the HIV-1 strain LAI. Conserved residues of gpl20 were altered to generate the HXBc2 gpl20 mutants. The interaction between the 20 mutants .and Fabs was investigated to examine binding specificity differences between the Fabs at greater resolution. The HXBc2 gpl20 mutants used in this assay had been previously characterized with respect to gpl60 precursor processing, gp120gp41 association, and CD4 binding ability tOlshevsky et al., J. Virol., 64: 5701-5707 Both wild type and mutant gpl20s were tested for their ability to bind a saturating concentration of each Fab.

The epitope determination with wild-type and mutant gpl20 was performed with HIV-1 envelope glycoproteins from culture supernatants of COS-1 cells transfected with plasmids expressing either wild-type or mutant gpl20 from the HXBc2 clone.

146 Microtiter wells were coated with the antibody D7324 (Aalto BioReagents; Dublin, Ireland) which binds to the conserved 15 amino acid sequence at the carboxy terminus of gpl20. The wild-type or .mutant gpl20 were thus captured onto the surface of microtiter wells by binding to the D7324 antibody.

A reference HIV-1 positive human serum pool at a 1:3000 dilution in 0.5% Tween 20 was assayed for binding to the wild-type and mutant gpl20s by incubating the serum pool with the immobilized gpl20. The bound antibody was detected by a second enzyme conjugated antibody. The reading obtained with the HIV-1 positive human serum pool, N=4, was used as the reference value for each mutant. The 15 Fabs of this invention were then assessed for binding to the wild-type and mutant gpl20s and the ratio of the Fab to reference serum was determined for each gpl20 mutant (Table The average ratio for the entire panel of Fabs was calculated and any -individual ratio deviating from the mean by less than 0.5 times was considered to indicate a gpl20 amino acid change that decreased Fab recognition, :..while those deviating by more than 2.0 times indicated.an amino acid change that enhanced Fab recognition. In this way, a map of mutations affecting the binding of the Fab to gpl20 was obtained for each clone essentially as previously described (Helseth et al., J. Virol.., 65:2119-2123 (1991) and Olshevsky et al., supra).

Table 9 Mutation Fab B3 B6 Bil B12 B13 B14 W/S 1.60 0.61 0.50 0.68 1.20 0.28 113 D/A 1.46 1.73 1.89 1.13 0.99 0.00 147 Mutation Fab 113 D/R 1.40 1.50 1.61 0.67 0.71 0.00 NO Vl/V2. 1.07 1.48 1.42 0.23 0 .86 1.68 NO Vl/V2/V3 2.05 1.48 1. 94 0.47 0.95 1.60 NO V3 1.88 1.64 1.92 0.46 1.08 1.72 183/184 PI/SG 0.82 0.73 0 .69 0.33 0.92 0.32 207 K/W 1.15 1.57 1.19 2.54 1.30 1.36 252 R/W 1.58 1.52 1.58 1.65 1.39 2.04 256 S/Y 0.64, 0.14 0 .33 0.82 1.15 0.00 257 T/R 0.08 0.59 0 .00 0.76 0.22 0.00 10 257 T/A 0.86 0.93 0 .75 0.99 0.68 0.40 257 T/G 0.91 0.70 1 .14 0.74 0.75 0.00 262 N/T 1.06 0.64 1.19 0.62 0.72 0.24 269 E/L 0.73 0.48 0.45 0.78 0.83 0.20 314 G/W 0.59 0.36 0.39 0. 65 0.71 0.28 ".15 356 N/I 0.67 0.66 0.39 0.92 0.80 0.52 368 D/R 0.19 0.18 0.00 0.04 0.00 0.00 368 D/T 0.28 0.20 0.00 0. 03 0.02 0.00 370 E/R 0.01 0.25 0.17 0.07 0.00 0.00 370 E/Q 0.25 0 89 0.58 0.46 0.14 0.00 384 Y/E 1.21 1.02 1.11 0.25 0.02 0.88 386 N/Q 0.88 0.59 0.31 1.05. 0.01 0.36 395 W/S 0.92 0.59 0.47 1.00 1.05 0.12 -427 W/S 1.57 1.11 1.53 0 .63 0.98 0.00 435 Y/S 1.93 1.16 1.58 1.41 1.24 2.04 450 T/N 0:62 0.48 0.58 0 .75 0.75 0.60 i" 148 Mutation Fab 457 D/A 0.62 0.39 0.44 0.28 0.62 0.20 457 D/R 0.84 0.55 0.92 0.32 0.58 0.56 470 P/L 0.80 0.64 0.72 0.72 0.18 0.24 475 M/S 0.06 1.02 0.33 1.50 1.39 0.92 477 D/V 0.50 0.09 0.00 0.07 0.52 0.00 o The general patterns observed are broadly similar to many CD4 site antibodies and of soluble CD4. Fab b12 is distinguished by its decreased binding to a mutant in which the VI and V2 loops are deleted. This may or may not be related to the enhanced neutralizing ability of Fab b12. However, it is clear that the VI and V2 loops and the V3 loop can affect antibody binding to the CD4 binding site either by direct contact or by transmitted *S conformational effects. Sensitivity to certain mutations in residues, particularly towards the C-terminus of gpl20, has previously been associated with CD4 binding site

U.

antibodies (Thali et al., J. Virol., 66:5636-5641 (1992) and Thali et al., J. Virol., 65:6188-6193 These mutations include residue 257 iil mutated from threonine to arginine (257 368 D/R, 370 E/R, 457 D/A and 477 D/V. Most of these mutations abrogate Fab binding or reduce it to low -levels consistent with the assignment of the recombinant Fabs in this assay as reacting with the CD4 site.

In a particular mutant of gpl20, the V1/V2 loop (residues 119-205) is completely removed.

This mutation enhances the binding of Fabs b6, bll, and b14 but significantly decreases the binding of Fab b12. Deletion of the V3 loop produces a more modest decrease in Fab b12 binding while generally h ,i :1 I

I

1-1 149 enhancing the binding of the other Fabs. The 314 G/W change in the V3 loop produces a decrease in binding of all the Fabs. This effect has been observed for other CD4 binding site antibodies (Moore and Sodroski, unpublished observations).

When the binding specificities of each Fab is -examined in detail, each Fab has a unique mutant binding profile. For example, Fab b14 binding is eliminated by the 113 D/A change whereas the binding of the other Fabs is unchanged or enhanced; Fab b3 and bli binding is reduced by the 475 M/S mutation but binding by the other Fabs is unchanged and the 370 E/Q change reduces binding of all the Fabs except for b6 and possibly bll. Fab b12 is 1 15 distinguished by its decreased binding to a mutant in which the V1 and V2 loops are deleted. This may or may not be related to the enhanced neutralizing ability of Fab b12 and will be the subject of further study. However, it is clear that the Vl and V2 loops and the V3 loop can affect antibody binding to the CD4 binding site either by direct contact or transmitted conformational effects.

The effects on Fab binding of a series of .point mutations in gpl20 afford the opportunity to 25 look more closely at recognition differences. The generalpatterns observed are broadly reminiscent of many CD4 site antibodies and of soluble CD4 itself. Fab b12 is distinguished by its decreased binding to a mutant in which the Vl and V2 loops are deleted. This may or may not be related to the enhanced neutralizing ability of Fab b12. It will be necessary to study a number of variants of Fab b12, which could be produced by chain shuffling or mutation, to answer this question. However, it is clear that the Vl and V2 loops and the V3 loop can affect antibody binding to the CD4 binding site either by direct contact or transmitted conformational effects.

P

150 6. Determination of the Relationship Between the Epitopes Recognized by the Fabs with HIV-1 Antigen Multimeric Complexes a. Comparison of Fab Epitopes with cp120 and cpl60 Expressed as Multimeric Complexes on the Surface of COS-1 Cells Given the lack.of correlation of Fab neutralization with binding parameters assessed using recombinant gpl20, the binding of Fabs b3, t b6, and b12 to COS-1 cells expressing the HXBc2 envelope glycoproteins gpl60 and gpl20 was compared. Fab b3, the poorest neutralizer, Fab b6, Salso a poor neutralizer, and Fab b12, the most effective neutralizer as determined in Example 15 were used in the assay. The envelope glycoproteins expressed by the COS-1 cells were gpl60, the precursor of.gpl20 and gp41, and the mature In this assay, different concentrations of Fab were incubated with radiolabeled COS-1 cells which '0 express gpl60 and gpl20 on their surface. The cells were then washed and lysed. The gpi20 and envelope glycoproteins bound to Fab were precipitated with goat anti-F(ab') 2 antibody and analyzed by protein gel electrophoresis, and shown in Figure 20. Since the amount of HIV-1 envelope glycoprotein expressed on the surface of transfected COS-1 cells is .small compared with the amount present intracellularly, after cell lysis, the bound Fab is presented with a large excess of both mature gpl20 and gpl60 precursor forms. The total amount of envelope glycoproteins precipitated thus provides an indication of the amount of Fab bound to the cell surface. Scanning densitometry profiles were derived from the autoradiographs and are expressed in arbitrary densitometric units.

Although the lack of saturation for Fabs b6 and b3 precludes a precise estimate of affinity, it is clear that Fab b3 exhibits a lower affinity for 151 the precursor gpl60 than either Fab b6 or b12.

When the binding of Fab b12 and b6 are compared, several differences are apparent. Assuming that Fab 6 achieves saturation at concentrations -slightly higher than 150 pg/ml, the estimated affinities of Fab b12 and b6 for the total population of envelope glycoproteins recognized.

differ only marginally. The most striking difference in the binding of Fab b12 and b6 to the multimeric envelope glycoprotein complex is the preferential detection of gpl20 relative to by Fab b12. Using densitometry to estimate amounts, it is seen from Figure 20 that Fab b12 immunoreacts with an amount of gpl20 that is at 15 least about 50 more than the gpl60 present in the Simmunoreaction admixture. The estimated affinities,.based on the Fab concentrations at which half-maximal binding to gpl2.0 is observed, are 3 x 107 M and <6 x 106 M- 1 for Fabs b12 and b6, respectively.

The binding of the Fabs to the multimeric *envelope glycoprotein complex on the transfected COS-1 cell surface provides some insights into the observed.differences in neutralization potency.

25 The binding of the most potent neutralizing Fab, Fab b12, achieves saturation at roughly 100 gg/ml, whereas neither of the less potent neutralizing Fabs achieves saturation even at 150 4g/ml. Fab b3 clearly exhibits a lower affinity for the cell surface envelope glycoprotein complex than do the other two Fabs tested, b12 and b6. The most striking difference in the binding of b12 and b6 to the multimeric envelope glycoprotein complex is the preferential precipitation of gpl20 relative to gpl60 by the bound Fab b12. In addition to these differences in gpi20 recognition, it appears that the overall number of cell surface envelope glycoproteins capable of being recognized by the 152 less neutralizing Fabs is greater than that seen for Fab bl2. These differences suggest that Fab b12 may recognize a more limited subset of envelope glycoprotein conformations and that these conformations are better approximated by the mature glycoprotein in the cell lysates. It is known that the gpl60 precursor assumes a.greater variety of conformations during the maturation process than does the fully folded gpl20 product (Thiriart, et al., J. Immunol., 143:1832-1836 (1989) and Fennie and Lasky, J. Virol., 63:639-646 (1989)). The enhanced neutralization ability of Fab b12 could reflect a higher affinity for a restricted gpl20 conformation present in the functionally relevant subset of envelope glycoprotein spikes. Such a functionally relevant group of envelope glycoproteins moieties probably represents a small subset of the total population, consistent with the low infectious fraction

S.

2 0 associated with HIV-1 and other retroviral virus preparations. One caveat to these observations is that the glycosylation of gpl20 expressed as a recombinant protein in baculovirus or on the: surface of COS-1 cells is likely to differ and this could affect binding of the Fabs of this invention.

However, no difference in the affinity for CD4 binding site antibodies between the two forms of has been observed previously using a range of antibodies (Moore and Sodroski, unpublished observations). In addition, these studies employed a molecular clone of HIV-1 and its extension to primary isolates will need to be studied further.

Fabs derived from combinatorial libraries may be viewed as "artificial". However, as shown here, the recognition properties of a set of antibodies directed to the CD4 site of gpl20 show many features in common with those derived by conventional means. They also show many features 153 in common with one another suggesting that, with the caveats inherent in the library approach (Barbas et al., J. Molec. Biol., 230:812-823 (1993) and Burton and Barbas, Nature, 359:782-783 (1992)), one individual produces several clearly distinct antibodies directed to a common structural feature, the CD4 binding site. This is in agreement with observations made on anti-CD4 binding site antibodies using anti-idiotype antibodies (Chamat et J. Immunol., 149:649-654 (1992) and Hariharan et al., J. Virol., 67:953-960 (1993)).

One advantage of producing several antibodies is that escape (at least in binding terms) is made more difficult. The only mutations in Table 9 which essentially eliminate the binding of all the antibodies also reduce CD4 binding ability.

*.The observations presented here have significance for vaccine development. The most effective vaccine may need to induce antibodies to the CD4 binding site with properties similar to those of.Fab b12. Given the data above, recombinant gpl20 offers no special qualities in this regard., Further, the Fab b12 type of antibody formed only about iO% (4/33 Fabs) of the cloned response of the library donor (Barbas et al., J.

Molec. Biol., 230:812-823 (1993)) and has not been described amongst the human'antibodies derived by other means suggesting it may be a minor component of typical responses. It is clearly of some interest for vaccine design to define more precisely the structure recognized by Fab b12.

7. Recognition of aP120 from Primary HIV-I Isolates by b12 IqGl in Vitro The ability of the b12 IgGl to recognize the molecule from HIV-l virus from 69 primary isolates was determined in an ELISA assay.

Recognition of the primary HIV-I virus isolate with 154 b12 IgG1 is indicative of the prevalence of the b12 epitope in the HIV-1 pandemic. To probe the occurrence of the b12 epitope in the HIV-1 pandemic, binding of the b12 IgG1 to gpl20 from 69 international isolates belonging to 6 different -clades was examined. Virus isolates assayed were obtained from the WHO, HMJFAMM, and NIAID.

Infectious culture supernatants containing virus and free gpl20 were treated with 1%(v/v) Nonidet-P40 (NP40) non-ionic detergent to provide a source of gpl20 (Moore et al., AIDS, 3:155-160 (1989)). Microplate wells (Immulon II, Dynatech, Ltd.) were first coated with sheep polyclonal antibody D7324. This antibody was raised to the peptide APTKAKRRVVQREKR, derived from the Cterminal 15 amino acids of the clade B IIIB HIV-1 viral isolate. Next, an appropriate volume of inactivated supernatant containing gpl20 was .diluted with a .buffer comprising tris-buffered

Z.

2 0 saline (TBS)/1% NP40/10% fetal calf serum (FCS) and a 100 Al aliquot added to the microplate wells for 2 hours at room temperature. Unbound gpl20 was removed by washing with TBS, and bound gpl20 was detected with CD4-IgG (1 pg/ml) or with b12 IgGl diluted in a buffer comprising TBS/2%(w/v) nonfat dry milk powder/20%(v/v)sheet serum (TMTSS) essentially as previously described (Moore et al., AIDS, 4:307-310 (1990)) and Moore et al., J.

:Virol., 68:469-473 (1994)). CD4-IgG is a fusion molecule which consists of CD4 and IgG. The CD4 portion binds to gpl20 and the IgG portion provides the means for detection of the CD4-IgG fusion molecule with labeled anti-IgG reagents. Bound antibody was then detected with an appropriate alkaline-phosphatase conjugated anti-IgG, followed by AMPAK (Dako Diagnostics). Absorbance was determined at 492 nm (OD 4 Each virus was tested against CD4-IgG in triplicate and against b12 IgGi 155 in duplicate. All OD 492 values were corrected for non-specific antibody binding in the absence of added gpl20 (buffer blank). The mean, blankcorrected OD 492 values for CD4-IgG and b12 IgGl were then calculated, and the OD4.2 ratios of b12 IgGI:CD4-IgG were determined. This normalization procedure enables allowance to be made for the different amounts of gpl20 captured onto the solid phase via antibody D7324 when comparing antibody reactivity with a panel of viruses. Binding ratios of 0.50 or greater were deemed to represent strong antibody reactivity; ratios from 0.25-0.50 were considered indicative of moderate reactivity; values of <0.25 were designated as representative of essentially negative monoclonal antibody reactivity.

As shown in Figure 22, b12 IgGI reacts with ;50% of clades A-D but only 1 of 12 isolates from clade E. Reactivity with clade B isolates from the 20 U.S.A. is approximately 8. Nucleic Acid Sequence Analysis Comparison Between HIV-1 Specific Monoclonal Antibody Fabs and the Corresponding Derived Amino Acid 25 Residue Sequence To explore the relationship between neutralizing and weakly or non-neutralizing Fabs, the variable domains of 32 clones expressing human Fabs, prepared in Example 2 including the 20 listed in Figure 6 for which neutralizing activity was assessed, were sequenced. In addition, the five gp41-specific Fabs were also sequenced.

Nucleic acid sequencing was performed on double-stranded DNA using Sequenase 1.0 (USB, Cleveland, OH) and the appropriate primers hybridizing to sequences in the Cgl domain (SEQGb GTCGTTGACCAGGCAGCCCAG 3' SEQ ID NO 49) or the Ck 156 domain (SEQKb 5' ATAGAAGTTGTTCAGCAGGCA 3' SEQ ID NO 50). Alternatively sequencing employed single stranded DNA and the T3 primer ATTAACCCTCACTAAAG SEQ ID NO 51) or one hybridizing to a sequence in the Ck domain (KEF GAATTCTAAACTAGCTAGTTCG 3' SEQ ID NO 52).

The amino acid residue sequences of the variable heavy and light chains derived from the nucleic acid sequences of the 32 clones are shown respectively in Figures 10 and 11.

Groupings are made on the basis of similarities in heavy chain sequences. Dots indicate identity with the first sequence in each section. The SEQ ID NOs are listed to the right of the corresponding derived heavy and light chain (VH from SEQ ID NO 53- 81 and VL from SEQ ID NO 82-113) amino acid residue sequences in the Figures themselves.

Alignment of derived sequences with one another and with the Genbank database made use of ""20 the MacVector suite of programs. For analysis of heavy chain CDR3 sequences as described by Sanz, J.

Immunol., 147:1720-1729 -(1991), the most nucleotide was considered to be the first nucleotide after codon 95 of the H chain variable region according to Kabat et al, Sequences of Proteins of Immunological Interest, US Dept. of I Health and Human Services, Washington, DC (1991).

The most 3' nucleotide was assigned to the last unidentified nucleotide before the sequence matched with the published germline JH genes. The CDR3 sequences were analyzed using the DNASTAR software.

Sequence comparisons were performed with both the ALIGN and COMPARE programs in order to determine the germline D gene which provided the best homology throughout. In a second step, the SEQCOMP program was used to find sequence identity of at least six nucleotides with either the coding strand or the reverse complement of germline D genes.

I. I- II I; IIj p I. I 157 The heavy and light chain sequences 'of the gp41-specific Fabs are shown in Figures 18 and 19, *respectively. The amino acid residue sequence of the CDR3 heavy chain exhibits the most variation between the Fabs than any other region of the 'variable domain.

a. Organization of Antibodies into Groups According to Heavy Chain Sequence VH and V 1 domains of 32 gp120 clones were *.,sequenced and the V. domains 'compared using .MacVector software. This analysis immediately established that a number of the 'clones, including 'those selected by panning against different antigens, are closely related to one another. The exception to this is the Fabs selected by panning against the V3 loop peptide which are not related to the Fabs selected by panning. against the gp12O/l60 antigens. Figures 10A and 103 show that *th .e VH sequences derived from gp12O0/16O panning can be organized into 7 groups. The broad features apparent from a comparison of amino acid sequences are discussed herein.

The relatedness of sequences within a group .varies considerably. For instance, in the group beginning with clone number b8 the amino acid sequences are very similar. Six clones were identical and the remainder showed a maximum of differences from the predominant sequence (the. EQ difference due to the 5' primer excluded) only one clone showed a single difference in the CDR3 region. The average discrepancy over all the sequences in this group from the predominant sequence is 1.1 amino acid residues/ variable *domain. This amount corresponds to the order of magnitude of discrepancies which could arise from the PCR. Sequencing of constant domains indicated a PCR error frequency of about I base change per 158 domain.

In contrast, in the group headed by clone b3, no two clones were absolutely identical. The average difference from the consensus group .'*sequence is 3.3 residues per sequence and Sdetermination for the CDR3 alone is 1.3.

Therefore, it seems likely that the heavy chains in this group are somatic variants of one another.

The group headed by clone 1 presents a third 10 pattern. Clones bl and b14 are identical as are clones b2 and B2. However, 23 amino acid differences exist between the two sets of clones.

S..Clones b24 and B30 are approximately equally well differentiated (13-25 differences) from either of these two sets of clones or one another. Still the CDR3 regions are very similar. A number of explanations can be suggested for this pattern: 1) all clones in this group originate from the same germline gene which has undergone extensive somatic 20 mutation, 2) cross-over events have occurred to essentially recombine different germline genes with the same DJ combination, 3) a "convergent evolution" process has led to the selection of different germline genes associated with the same DJ combination.

b. Secuences of the VL Domains from the lD120 Binders The VL sequences of the Fabs were organized into the groups defined in Figures and 10B are shown in Figures 11A and 11B.

Immediately apparent was the extensive chain promiscuity as evidenced by the pairing of different light chains with the same or a very similar heavy chain with retention of antigen binding capability and indeed, for the most part, antigen affinity as compared with Figures 10A and This promiscuity can be explored further by I; 1- 159 reference to the groups considered above.

The clone b8 group, in which the heavy chain members were identical or very similar, also produced 4 light chains which are identical or very similar (less than 3 amino acid differences).

Therefore a predominant heavy-light chain -combination can be described for this group. One member (clone b8) had the same or very closely related VL gene but appeared to use a different Jk gene. Two other members (clones B8 and b18) were more distantly related to the major sequence (7-12 differences). Two further clones (bl3 and B26) used.a.Vk gene from a different family, Vk3 compared to Vkl, and therefore were unrelated to the major sequence.

The clone b3 group, suggested to contain somatic variants of a single heavy chain, showed considerable light chain diversity with no two members being closely related to one another.

Vk3-Jk2 combinations predominated but Vk3-Jk3 and Vkl-Jk3 combinations also occurred.

On the other hand, in the clone bl group 'i evidence existed for the heavy chains being more i choosy about their light chain partner.. Thus, 25 closely related heavy chains appeared to be paired with related light chains. The identical heavy chain pairs (bl and b14; b2 and B2) had very similar light chains (2 and 4 amino acid differences respectively) whereas the distinct heavy chains (b24 and B30) had distinct light chains which were unrelated to one another or the other group members. The clone 4 group provides another example of this phenomenon in that 4 closely related heavy chains were paired with 3 closely related light chains (a predominant heavy-light chain combination), except for the clone b7 light chain that was distinct.

7 160 In summary, the heavy chain (VH) sequences was organized into 7 groups where each member of a group has an identical or very similar CDR3 region with a limited number of differences elsewhere.

'When the light chains (VL) were constrained into the -groupings defined by-their heavy chain partners, considerable light chain sequence variation was observed. This phenomenon of chain promiscuity has been observed previously and can be appreciated by :.10 reference to Figures 11A and 1113. Marked **:neutralizing ability was confined to two groups of sequences. The first group consisted of Fabs 4, 7, 12 and 21 which have very similar heavy and light 0% chains. The second group 'consisted of .Fabs 13, 8, 18, 22 and 27. only.Fab 13 showed marked *neutralizing ability, although the others showed some weaker activity. -Interestingly in this group Fab 13 did have a light chain distinct from the other members of the group.

.0009.- Shufflingr of the Heavy and Light Chain of a Single 'Clone Against the Library 1. 0. To further explore possible functional heavy-light chain combinations, the heavy chain of clone b12 (also referred to as Fab 12 for the corresponding soluble Fab preparation) *shown in Figures 10A and 10B was recombined with the original light chain library prepared in Example 2 to cdnstructa anew library H12-LCn. In addition, the b12 light chain was recombined with the original heavy chain library to construct a library H-n-Ll2. These two libraries were taken through 3 rounds of panning against gp12O (IIIB) as described in Example 2b5) The Fabs expressed from the resultant immunoreactant clones were analyzed as described in Example 3 above. Clone b12 was chosen as this Fab neutralized HIV-l in vitro as shown in Example 3.

7_1..

161 To accomplish the preparation of a shuffled library from the Fd gene of clone b12 with the original light chain library, the b12 heavy chain was- first subcloned into a tetanus toxoid binding clone expressed in pComb2-3. The light chain library was then cloned into-this construction to -ive a library of 1 x 107 members. The subcloning step was used to avoid contamination with and over-representation of the original light chain. A similar procedure was adopted for shuffling Of heavy chains against the light chain from clone b12 to give a library of 3 x 106 members. Cloning and panning procedures were carried out as described 0 above for the original library.

Eleven light chains which recombined with the b12 heavy chain and bound gpl20 by panning were randomly chosen for subsequent competition ELISA and sequence analysis. The apparent affinities of these shuffled. combinations were similar with an 20 IC50 of approximately 10.8 to 10-' M. The sequences were organized where a set of 3 were very similar to the original b12 light chain and the other 8 showing many differences from the original with some sub-grouping possible.

0 The sequences of the light chains which bound to the b12.heavy chain clone are shown in Figures 12A and 12B. The sequences are compared to the sequence for the original light chain from.clone b12. The light chains are identified by numbers which do not correspond to.the original light chain clones; the assigned numbers of the newly selected clones having new light chains are thus arbitrary.

The sequences of these light chains are also listed in the Sequence Listing from SEQ ID NO 114 to 122.

35 Some light chain sequences are identical. In addition to immunoreactivity with gpl20, the new Fabs isolated from these shuffled clones were tested in the syncytia assay for neutralization of 162 HIV-1 infection as described in Example 3. Four shuffled monoclonal Fab antibodies, each having the heavy chain from clone b12, a known HIV-1 neutralizing clone, and new light chains designated .L28, L25, L26 and L22, all exhibited approximately 60* neutralization in a syncytia assay with 0.4 ,ug/ml purified Fab. This effect was equivalent to that obtained with the original clone b12 heavy and light chain pair. Maximum neutralization of approximately 8011 was obtained with the.H12/L28 and H12/L25 Fabs at 0.7 jg/ml which was equivalent to that seen with the original clone b12 heavy and light pair. The neutralization resulting from the H12/L22 and H12/L26 Fabs plateaued at 60* with Fab concentrations of 0.4 Ag/ml up to 1.0 Ag/ml. Thus, *.in addition to the gpl20 immunoreactive and HIV neutralizing Fabs obtained in the original library prepared as described in Example 2, by shuffling a known neutralizing heavy chain with a library of light chains, new HIV-I neutralizing Fab monoclonal 0900 antibodies have been obtained.

Ten heavy chains which recombined with the b12 light chain were also randomly chosen. One was very similar to the original bi2 heavy chain but the others have many differences. Nevertheless, the V-D and D-J junctions were essentially identical indicating the clones had probably arisen from the same rearranged B-cell clone by somatic modification. Competition ELISA failed to reveal any clear difference in affinity between the variants selected from those originally analyzed.

The sequences of the heavy chains which bound to the b12 light chain clone are shown in Figures 13A and 13B. The sequences are compared to the sequence for the original heavy chain from clone b12. The heavy chains are identified by numbers which do not correspond to the original light chain clones; the assigned numbers of the newly selecte dN.

163 clones having new heavy chains are thus arbitrary.

The sequences of these light chains are also listed in the Sequence Listing from SEQ ID NO 123 to 132.

Some light chain sequences are identical. In addition to immunoreactivity with gpl20, the new clones were tested in the syncytia assay for neutralization of HIV-I infection as described in Example 3. Two shuffled monoclonal Fab antibodies, each having the light chain from clone b12, a known HIV-I neutralizing clone, and new heavy chains designated H2 and.H14, exhibited approximately neutralization in a syncytia assay with 1.0 and jLg/ml purified Fab, respectively. This effect was equivalent to that obtained with the original clone 15 b12 heavy and light chain pair at a concentration i of 2 Ag/ml. Maximum neutralization of approximately 50% was obtained with the Fab having the new H14 chain at 1.0 4g/ml compared to neutralization with 0.7 jig/ml with the original clone b12 heavy and light pair. Thus, in addition to the gpl20 immunoreactive and HIV neutralizing Fabs obtained in the original library prepared as described in Example 2, by shuffling a known neutralizing light chain with a library of heavy chains, new HIV-1 neutralizing Fab monoclonal antibodies have been obtained.

Thus, this shuffling process revealed many more heavy and light chain partners that bound to that were equal in affinity to those obtained from the original library prepared in Example 2.

With this. approach, additional HIV-1 neutralizing antibodies can easily be obtained over those present in an original library. The complexity of the clones arising from the heavy chain shuffling 35 also suggests that this approach may be used to map the course of somatic diversification.

Combinatorial libraries randomly recombine heavy and light chains so to what extent antibodies 164 derived from such libraries represent those produced in a response in vivo can be determined.

In principle, a heavy-light chain combination binding antigen could arise fortuitously, i.e., neither chain is involved in binding antigen in -vivo but the combination does bind antigen in vitro.

The available data suggests, however, that heavy chains, from immune libraries, involved in :o.i01 binding antigen tightly in vitro arise from sees o6° antigen-specific clones in vivo. First, studies have generally failed to identify high-affinity "sso*binders in non-immunized IgG libraries. See, Persson et al. Proc. Natl. Acad. Sci., USA, 88:2432-2436 (1991) and Marks et al. Eur. J.

Immunol.., 21:985-991 (1991).

Further, as described above, gpl20 binders were not observed in panning a bone marrow IgG library from an HIV seronegative donor against gpl20. Second, heavy chains associated with binders from immunized libraries were'typically at relatively high frequency in the library indicating they were strongly represented in the mRNA isolated o from immunized animals. See, Caton et al., Proc.

N:..Natl. Acad. Sci. USA, 87:6450-6454 (1990) and Persson et supra. Third, heavy chains from immunized libraries appeared to dictate specificity when recombined with various unrelated light chains as described in Example 10. Fourth, the isolation of intraclonal heavy chain variants as here indicated that an active antibody response was cloned. Thus, the shuffling of a known heavy chain with a light chain binder and vice versa is preferred for use in this invention as new neutralizing Fabs can be obtained beyond those generated in vivo.

Heavy chain promiscuity, the ability of a heavy chain to pair with different light chains 165 with retention of antigen affinity, presents serious problems for identifying in viva light chain partners. This applies not only to the strict definition of partners as having arisen from the same B-cell but also to one which would encompass somatic variants of either partner. The -existence of predominant heavy-light chain combinations, particularly involving intraclonal light chain variants, suggests that the light chains concerned are well represented in the library and probably are associated with antigen binding in viva. However, promiscuity means that, although some combinations probably do occur in *..vivo, one cannot be ce rtain that one is not shuffling immune partner chains in the recombination. For instance, the occurrence of a virtuallyidentical light chain (b6, B20) in 2 out of 33 clones suggests that it is probably -over-represented in the library consistent with an in viva involvement in antigen-stimulated clones.

However,, there is no way of knowing whether the in viva partner of the light chain is the b6 or heavy chain or indeed another heavy chain arising from a stimulated clone.

The light chains arising from the *.combinatorial library may not be those employed in v iva. Nevertheless it is interesting to note that some heavy chains appear relatively choosy about light chain-partner whereas others appear almost indifferent. -This observation needs tobe tempered by the finding that apparently choosy heavy chains from this analysis will accept diverse light chains with maintenance of antigen binding in a binary plasmid systemwhere pairings are forced as shown 35 below it Example 11 rather than selected in a competitive situation.

Two reports compare heavy-light chain combinations' arising from combinatorial libraries .4 166 and hybridomas in immunized mice. The library approach begins with mRNA and is therefore probably reflecting plasma cell populations. In contrast, hybridomas are thought to reflect activated but not terminally differentiated B cell populations and EBV transformation to reflect resting B cell populations.

Whatever the arguments about light chain authenticity, the heavy chains of Figures 10A and 10B present many features of interest. The most frequently used heavy chain is of the clone b8 type. It could be argued that this usage simply represents bias in PCR amplification. However, the occurrence of approximately equal numbers of clones in this group amplified by VHla and VH3a primers argues against this notion. Furthermore, the existence of intraclonal variants in some groups indicates that one is at least sampling different genes from the initial library.

The antibodies cloned here do bear qualitative relationship with the polyclonal antibodies present in the serum of the asymptomatic donor. The titer of anti-gpl20 (IIIB) antibodies was approximately 1:3000, with greater than 50% of the reactivity being inhibited by CD4 or .a cocktail of Fabs from.

clones 12, 13 and 14. The titer of anti-gp120 (SF2) antibodies was approximately 1:800. Further, the titer of serum against the short constrained V3 loop peptide was 1:500 and against the full length MN V3 loop peptide was only i:300. The importance of "anti-CD4 site antibodies" seems general in donors with longer term HIV infection in that the cocktail of Fabs 12, 13 and 14 was able to inhibit binding of a large fraction of serum antibody reactivity with gp120 (IIIB) in 26 of 28 donors tested.

The ability of Fabs to neutralize viruses has been a controversial area. One of the problems has C .t 7W-r-~.

7 167 beeni that Fabs are classically generated-by papain digestion o f IgG. If the Fab, as is often. the case, shows reduced activity relative to the parent IgG then it may be difficult to rule out IgG contamination in the Fab preparation. Recombinant -Fabs, however, as shown herein definitively .neutralize virus.

The mechanism of neutralization of HIV-l appears to neither require virion aggregation nor gpl2O cross-linking. In addition, there is .no correlation with blocking of the CD4-gpl2O interaction to neutralization. The existence of the cloned neutralizing Fabs of this invention should allow the molecular features that confer neutralizing potential to be explored. For instance, in the case of the group of clones containing Fab 13, the unique character of the light chain of that neutralizing clone suggests that chain shuffling experiments in which the 13 light chain was recombined with the other heavy chains in'that group, might be revealing. Heavy chains paired with two dissimilar light chains have been shown to retain antigen affinity but exhibit al tered fine specificity as shown in Example 11..

The observation here of a large. number of Fabs with only a limited number being strongly neutralizing may have important consequences. if the pattern is repeated for whole antibodies then it would seem that much of the gpl2O structure may -be in a sense a "decoy", the immune system may invest considerable effort in producing antibodies of high affinity but limited anti-viral function. To exacerbate the situation the ineffective antibodies may bind to gpl 2 O and inhibit the binding of strongly neutralizing antibodies. This has obvious consequences for vaccination which should be primarily designed to elicit neutralizing antibodies of this invention.

-74 71- 168 Shuffling of Selected Heavy and Light Chain DNA Sequences of a Combinatorial Library in a Binary Plasmid System A binary system of replicon-compatible .plasmids has been developed to test the potential -'for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. The efficiency of the system is demonstrated for the combinatorial library of this invention derived from the bone marrow library of an asymptomatic HIV 0: donor.

a. Construction of the Binary Plasmid System The binary plasmids pTACO1H and pTC01 for use in this invention contain the pelB leader S: region and multiple cloning sites from Lambda Hc2 "and Lambda Lc3, respectively, and the set of replicon-compatible expression vectors pFL281 and pFL261. Both pFL281 and pFL261 have been described by Larimer et al., Prot. Eng., 3:227-231 (1990), the disclosure of which is hereby incorporated by reference. The nucleotide sequences of pFL261 and.

pFL281 are in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers M29363 and M68946. The plasmid pFL281 is based on the plasmid pFL260 also described by Larimer et al., supra, and having the accession number M29362.

The only. distinction between the plasmids pFL260 and pFl281 is that pFL281 lacks a 60 bp sequence of pFL260 between the Eag I site and the Xma III site resulting in the loss of one of the two BamH I sites. This deletion is necessary to allow for cloning of the BamH I Hc2 fragment into the expression vector as described herein.

The replicon-compatible expression vectors share three common elements: the fl single-stranded DNA page intergenic IG regions; s 169 (ii) the tightly regulated tac promoter and lac operator; and (iii) an rbs-ATG region with specific cloning sites. The plasmid vectors differ in their antibiotic resistance markers and plasmid replicons: pFL261 carries a gene encoding chloramphenicol acetyltransferase (cat), conferring chloramphenicol resistance,a nd the pl5A replicon; pFL281 carries a gene encoding beta-lactamase (bla), conferring ampicillin resistance, and the ColEl replicon (ori) from pMB1. The pl5A and ColEl replicons.permit the coincident maintenance of both plasmids in the same E. coli host.

The Hc2 and Lc2 vectors prepared in Examples la2) and la3), respectively, were converted into the plasmid form using standard methods familiar to one of ordinary skill in the art and as described by Sambrook et al., Molecular Clonin: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York (1989) and subsequently digested with Xho I-Spe I (pHc2) and Sac I-Xba I for (pLc2) The synthetic linkers for insertion into the digested pHc2 and Lc2 plasmids were prepared by American Synthesis. The linkers were i: inserted to increase the distance between cloning sites so as to increase the effectiveness of the digestions. The 5' and 3' linkers for preparing the double-stranded linker insert into pHc2 were TCGAGGGTCGGTCGGTCTCTAGACGGTCGGTCGGTCA 3' (SEQ ID NO 133) and 5' CTAGTGACCGACCGACCGTCTAGAGACCGACCGACCC 3' (SEQ ID NO 134), respectively. The 5' and 3' linkers for preparing the double-stranded linker insert into pLc2 were CGGTCGGTCGGTCCTCGAGGGTCGGTCGGTCT 3' (SEQ ID NO 135) and 5' CTAGAGACCGACCGACCCTCGAGGACCGACCGACCGAGCT 3' (SEQ ID NO 136), respectively. The pairs of linker oligonucleotides were separately ligated to theiri': respective digested, calf intestinal phosphatasetreated vectors.

-LI~U 170 Subsequently, the multiple cloning sites of pHc2 and pLc2 were transferred into the expression vectors, pFL281 and pFL261, respectively. To accomplish this process, the multiple cloning regions of both Lc2 and Hc2 were separately amplified by PCR as described by Gram et al., Proc.

Natl. Acad. Sci., USA, 89:3576-3580 (1992) and as described in Example 2b using Vent Polymerase (New England Biolabs) according to the manufacturer's recommendations. The forward primer, CAAGGAGACAGGATCCATGAAATAC 3' (SEQ ID NO 137) was designed to provide a flush fusion of the pelB leader sequence to the ribosome binding sites of the cloning vectors pFL261 and pFL281 via its 15 internal BamH I site indicated by the underlined nucleotides. The reverse primer AGGGCGAATTGGATCCCGGGCCCCC 3' (SEQ ID NO 138) was designed to anneal downstream of the region of interest in the parent vector of pHc2/pLc2 and 20 create a second BamH I site. The resultant Hc2 and Lc2 PCR amplification products were then digested ii with BamH I to provide for BamH I overhangs for subsequent ligation into BamH I linearized pFL281 and pFL261 vectors, respectively. The resulting light chain vector containing the Lc2 insert, designated pTC01, was used in this form, whereas the heavy chain vector was further modified with a:: histidine tail to allow purification of Fab fragments by immobilized metal affinity chromatography as described by Skerra et al., Bio/Technoloy, 9:273-278 (1991). For this purpose, the synthetic linker oligonucleotides, respectively the 5' and 3' linkers, CTAGTCATCATCATCATCATTAAGCTAGC 3' (SEQ ID NO 139) and 5' CTAGGCTAGCTTAATGATGATGATGATGA '3 (SEQ ID NO 140) was inserted into the Spe I site, in effect removing the decapeptide tag sequence to generate the heavy chain vector designated as pTAC01H. The 171 expression of Fab fragment in all subsequent cloning experiments was suppressed by adding 1% glucose to all media and plates.

b. Construction of Expression Plasmids For expression of the light chain -variable domain, pTC01 prepared above was first digested with Sac I and Xba I; individual light chain inserts were then obtained by separately digesting 22 of the pComb2-3 plasmids prepared and screened as described in Example 2 and listed in Figure 7 that bind to gpl20 with the same combination of enzymes and isolating the 0.7 kb fragment using low melting point agarose gel electrophoresis followed by b-agarose digestion.

For the chain-shuffling experiments, the following representative members of each of the seven groups shown in Figure 7 were chosen: bll; b6; b4-b12-b7-b21; b3; s8; bl-b14-b24; bl3-b22-B26-b8-bl8-b27-B8-B 3 5-s 4 and one loop peptide-binding clone, p35. The different groups are indicated by semicolon separations while members of the same group are dashed. The.

Sresultant isolated light chains were separately ligated into PTCO1 overnight at 16 0 C under standard ee ""conditions using a 5:1 molar insert-to-vector ratio to form 21 light chain pTCO1 expression vectors.

For expression of the heavy-.chain variable domain, pTAC01H prepared above was first digested with Xho I and Spe I; heavy chain inserts were then obtained by separately.PCR amplification reactions of the pComb2-3-plasmids from which light chain inserts were obtained. PCR was used to isolate the heavy chain inserts instead of restriction digestion in order to obtain heavy chain without the cpIII gene anchor sequence in the vector. For the PCR reaction, the respective 5'.and 3' primers, CAGGTGCAGCTCGAGCAGTCTGGG 31 (VHla) (SEQ ID NO 42) 172 and 5' GCATGTACTAGTTTTGTCACAAGATTTGGG 3' (CGlz) (SEQ ID NO 44) were used to amplify the region corresponding to the heavy chain as described in Examples 2al) and 2a2). The resultant PCR products .were purified by low-melting point electrophoresis, -digested with Xho I and Spe I, re-purified, and separately ligated to the similarly prepared heavy chain pTACO1H vector using a 1:2 molar vector-toinsert ratio to form 21 heavy chain pTACO1H expression vectors.

c. Co-transformation of Binary Plasmids CaCl 2 -competent XL1-Blue cells (Stratagene; recAl, endAl, gyrA96, thi, hsdR17, supE44, relA, lac, F' p, lacF' lacI, ZDM15, i' Tnl0(tet")}) were prepared and transformed with approximately 0.5 pg purified DNA of each .plasmid in directed crosses of each of the 20 light chain vectors with each of the 20 heavy chain vectors.

20 The presence of both plasmids and the episome was selected for by plating transformants on triple-antibiotic agar plates.(100 yg/ml carbenicillin, 30 pig/ml chloramphenicol, 10 Ig/ml tetracycline, 32 g/l LB agar) containing 1% glucose.

A binary plasmid system consisting of two replicon-compatible plasmids was constructed as shown in 14. The pTACO1H heavy chain vector i schematic is shown in Figure 14A and the pTCO1 light chain vector schematic is shown in Figure 14B. Both expression vectors feature similar cloning sites including pel B leader sequences fused to the ribosome binding sites and the tac promoters via BamH I sites as shown in Figures and 15B. The nucleotide sequences of the multiple cloning sites along with the tac promoter, ribosome binding sites (rbs) and the underlined relevant restriction sites for the light chain vector, 173 pTCOl, and heavy chain vector, pTAC01H, are respectively shown in Figure 15A and Figure The sequences are also listed in the Sequence Listing as described in the Brief Description of the Drawings. The heavy chain vector pTAC01H also contains a (His)s-tail to allow purification of the recombinant Fab fragments by immobilized metal affinity chromatography. The presence of both plasmids in the same bacterial cell is selected for by the presence of both antibiotics in the media.

Expression is partially suppressed during growth. by addition of glucose and induced by the addition of IPTG at room temperature. Under these conditions, both plasmids are stable within the cell and "15 support .expression of the Fab fragment as assayed by ELISA using goat anti-human kappa and goat anti-human IgG1 antibodies.

d. Preparation of Recombinant Fab Fragments Bacterial cultures for determination of antigen-binding activity were grown in 96 well-tissue culture plates (Costar #3596). 250 pil Superbroth [SB had the following ingredients per liter: 10 g 3-(N-morpholino) propanesulfonic acid, 25 30 g tryptone, 20 g yeast extract at pH 7.0 at 250C) containing 30 pg/ml chloramphenicol, 100 ig/ml carbenicillin, and 1% glucose were admixed per well and inoculated with a single double-transformant prepared in Example lic above.

The inoculated plates were then .maintained with moderate shaking (200 rpm) on a horizontal shaker for 7-9 hours at 37 0 C, until the A 550 so was approximately 1-1.5. The cells were collected by centrifugation of the microtiter plate (1,500 X g for 30 minutes at 40C), the supernatants were discarded, and the cells were resuspended and induced overnight at room temperature in fresh media containing 1 mM IPTG, but no glucose. Cells 174 were harvested by centrifugation, resuspended in 175 p1 PBS (10 mM sodium phosphate, 160 mM NaCi at pH 7.4 at 25 0 C) containing 34 pg/ml phenylmethylsulfonyl fluoride (PMSF) and 1.5% (w/v) streptomycin sulfate, and lysed by 3 freeze-thaw cycles between -80 0 C and 37 0 C. The resultant crude extracts were partially cleared by centrifugation as above before analysis by antigen-binding ELISA.

e. Assay and Determination of Relative Affinities Relative affinities were determined as described in Example 2b6) after coating wells with 15 0.1 pg of antigen. The selected antigens included tetanus toxoid and recombinant gpl20 (strain IIIB) and gpl20 (strain SF2) For each antigen, a negative control extract of XL1-Blue cells :i i i: co-transformed with pTC01 and pTAC01H was tested to determine./whether other components in E. coli had.

any affinity for the antigens in the assay. Each extract was assayed for BSA-binding activity and S. BSA-positive clones were considered negative. All possible single-transformants expressing one chain only were prepared as described for the double-transformants and were found to have no affinity for any of the antigens used. Because of the nature of the assay, whether this was due to a lack of binding by the individual chains itself or due to a lack of expression or folding could not be determined.

f. Results of Direct Crosses of Heavy and Light Chains within a Set of pl20/qpl60 Binding Antibodies The Fab fragments derived from the bone marrow of the same asymptomatic HIV donor but panned against gpl20 (IIIB), gpl60 (IIIB), and iii~ -~l 175 (SF2), were assigned to one of seven groups based on the amino acid sequences of the CDR3 of their heavy chains as described in Example 9. From the same library, antibodies to the constrained hypervariable v3-loop-like peptide

JSISIGPGRAFYTGZC

(SEQ ID NO 141) were isolated. For the -chain-shuffling experiments, the following representative members of each of the seven groups shown in Figure 7 were chosen: bll; b6; b4-b12-b7b21; b3; s8; bl-b14-b24; bl3-b22-B26-b8-bl8-b27-B 8 B35-s4; and one loop peptide-binding clone, p 3 5 •Clones b4, b7, b12, and b21.showed neutralization activity against HIV when monitoring inhibition of infection by syncytia formation and clones b13, b12, and b4 when monitoring p24 production as shown in Example 3. Light and heavy chains were cloned from the original constructs and cotransformed in all possible binary combinations into XL1-Blue cells as described above.

The results of the complete cross are shown in Figure 16. As is to be expected, identical chains derived from different Fab fragments had similar coo binding properties bl8HC, b27HC, B8HC, s4HC. The crosses of the original heavy chains with the original light chains in each case clearly recapitulated binding activity. Minor differences existed between some heavy chains with identical variable domain sequences, b4 and b12 (constant domains were not sequenced for any of the constructs). .The exception is b8HC, which was.

identical in its variable domain to bl8HC, b27HC, B8HC, B35HC, s4HC, yet shows more cross reactivity.

Presumably, this is due to differences in expression levels in the cell or differences in the constant domain sequences. Clear differences existed between heavy chains in their tendency to accept different light chains and still bind antigen, but even the least promiscuous heavy chain 5 1 176 in the set panned against gpl20 (IIIB), b1HC, still did so in 43% of its crosses. On the other side of the spectrum, 5 heavy chains, b11HC, b6HC, bl2HC,.

b7HC, and b8HC, crossed productively with all light ichains in this set. For the heavy chain crosses examined in detail (all of s4HC, B35HC, B26HC; most of b12HC, bl2HC), no significant differences in apparent binding affinity were found between Fab fragments using the same heavy chain but different light chains as shown in Figure 17 where the IC 50 oo o from competition with soluble gp!20 (IIIB) was approximately 108.M.

Within the original seven groups that were established according to the sequence of the CDR3 of the heavy chains and that are indicated by horizontal and vertical lines in Figure 16, complete promiscuity was present, heavy and light chains within these CDR3-determined groups were completely promiscuous with each other.

However, there was a lack of promiscuity between other groups, between blHC-b24HC and bl3LC-s4LC. In the analysis of these sequence-based groups, the protein antigen against which the phage display library was panned was not 0 *25 a critical factor. The exception to this case was the cross of p35HC with all light chains; the only cross that bound either to gpl20 (SF2 strain) or the original antigen, the loop peptide, was the cross containing the original heavy and light -chains.

Unlike the heavy chains, no light chains crossed productively with all heavy chains nor were any distinguishable from the other light chains by unusually low promiscuity.

In the neutralization assays performed as described in Example 3, the directed cross.

resulting from the pairing of the heavy chain from clone b12 with the light chain from clone b21, was 177 effective at neutralizing HIV-1.

g. Interantiqenic Crosses of Heavy and Liqht Chains To determine whether conclusions derived from the crosses between high affinity Fab fragments originating from the same library can be extended to unrelated libraries, a non-related gammalk-Fab fragment (P3-13) specific for tetanus toxoid from a different donor was chosen for a new set of crosses [clone 3 in Persson et al., Proc7 Natl. Acad. Sci., USA, 88:2432-2436 (1991)].

Extracts were probed with tetanus toxoid or with (IIIB) The data confirm the results from the gp120 cross experiment in that the binding activity towards the antigen was determined by the heavy chain. The heavy chain of clone P3-13 paired .0 with the light chains b4, b12, b21, and b14 to yield an Fab fragment with an affinity towards tetanus toxoid; the light chain of P3-13 paired with the heavy chains of b3, b6, bil, and b14 to yield an Fab fragment with an affinity towards (IIIB). None of the light chains originating from the gpl2o binders was able to confer specificity in combination with the P3-13 heavy chain..

Similarly' the P3-13 light chain was unable to generate tetanus toxoid specificity in combination with any of the heavy-chains originating from the gpl20 binders, confirming the dominance of the heavy chain in the antibody-antigen interaction.

Interestingly, all three light chains that showed a strong signal against tetanus toxoid (b4, b12, b21) were members of the same group when sorted by the CDR3's of their original heavy chains. As might be expected from crosses between unrelated libraries, not only was there a lower degree of promiscuity, chains paired productively with far fewer 7 7 777VT& A~.

178 complementary chains, but the range of apparent affinity constants determined by competition ELISA was much broader (6.3 X 106 6.3 X 10- 8 The 'replacement of the original P3-13 light chain in o 5 .the P3-13 Fab fragment with another light chain "'-lowered the affinity of the Fab towards tetanus toxoid 10 to 100-fold (from 6.3 X 10- 8 M to 6.3 X 10-6 In the crosses of the light chain of P3-13 with all the heavy chains of the HIV pannings, the productive crosses had similar affinities to (IIIB) (2.5 X 107- 6.3 X 10- 7 with the exception of bl4HC/P3-13LC, whose signal was too weak for a definite determination of the apparent binding Iconstant. These affinities were approximately five-fold lower than those of the gpl20-heavy.. h'li chains with their original light chains.

Thus, the results show that chain shuffling is yet another maneuver allowed in vitro but not in vivo which can be expected to help extend antibody diversity beyond that of Nature.- The overriding feature of the binary system of this invention is its ability to create large numbers (several hundred) of directed crosses between characterized light and heavy chains without the need for recloning individual chains for each cross after the initial vector construction. When used in combination with the phage-display method and biological assays, it allows the rapid analysis of the most interesting subset of the pool of antigen-binding clones by chain shuffling, with the' aim of finding biologically or chemically active antibodies. For the set of antigens studied here, most heavy chains recombined with a number of light chains to yield an antigen-binding Fab fragment.

These results have important implications for the diversity of combinatorial antibody libraries.

While it is not possible to predict reliably the original in vivo combinations of light and heavy

A~

179 chains due to the surprising promiscuity of individual chains, recombinant antibody libraries take advantage of the fact that even distantly related Fabs against the same antigen can recombine in vitro to give chain combinations not found in vivo. In fact, after the identification of a certain number of antibodies that have been shown to possess some biological or chemical activity, it may be better to shuffle their individual chains in 0 a directed fashion than to continue sampling' randomly from the same pool of binders. By extension, the promiscuity observed in this system "':'.indicates that in libraries constructed using degenerate, chemically synthesized oligonucleotides, there should be considerable e -flexibility in which separate synthetic heavy chains can pair with separate synthetic light chains to generate separate antigen-binding Fab fragments. The diversity of combinatorial libraries coupled with chain-shuffling should allow wide exploration of three dimensional space thereby solving the problem of how to approximate molecules in the ternary complex of antibody, substrate and cofactor.

11. Denosit of Materials The following cell lines have been deposited onSeptember 30, 1992, with the American Type Culture Collection (ATCC), 1301 Parklawn Drive, Rockville, MD, USA: Cell Line ATCC Accession No.

E. coli MT11 ATCC 69078 E. coli MT12 ATCC 69079 E. coli MT13 ATCC 69080 The deposits listed above, MT11, MT12 and MT13 are bacterial cells coli) containing the 180 expression vector pComb2-3 for the respective expression of the Fabs designated bl (clone bil), b12 (clone b12) and b13 (clone b13) prepared in Example*2b. The sequences of the heavy and light .:.5.chain variable domains are listed in Figures and l0B and 11A and 11B, respectively. This deposit was made with the ATCC under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty).' This assures maintenance of a viable culture for 30 years from the date of deposit. The organisms will be made available by ATCC under the terms of the Budapest *15 Treaty which assures permanent and unrestricted availability-of the progeny of-the culture to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, ;.and assures availability of the progeny to one determined by the U.S. Commissioner of. Patents and Trademarks to be entitled thereto according to U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular .reference to .886 OG 638) The assignee of the present application has Agreed that if the culture deposit should die or be lost or destroyed when cultivated under suitable conditions, it will be promptly replaced on notification with a viable specimen of the same culture. Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present S- 181 invention is not to be limited in scope by the cell lines deposited, since the deposited embodiment is intended as a single illustration of one aspect of the invention and any cell lines that are functionally equivalent are within the scope of this invention. The deposit of material does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustration that it represents.

Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from :the foregoing description and fall within the scope of the appended claims.

el In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprising" is used in the sense of "including", i.e. the features specified may be associated with further features in various embodiments of the invention.

It is to be understood that a reference heiein to a Set.

prior art document does not constitute an admission that document forms part of the common general knowledge in the art in Australia or in any other country.

R I .of.

~Iv RA% 182 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: THE SCRIPPS RESEARCH INSTITUTE STREET: 10666 North Torrey Pines Road CITY: La Jolla STATE: CA COUNTRY: USA POSTAL CODE (ZIP): 92037 TELEPHONE: 619-554-2937 TELEFAX: 619-554-6312 (ii) TITLE OF INVENTION: HUMAN NEUTRALIZING MONOCLONAL ANTIBODIES TO HUMAN IMMUNODEFICIENCY VIRUS (iii) NUMBER OF SEQUENCES: 170 e (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version /1.25 (EPO) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US FILING DATE: 11-JUL-1995 (vi) PRIOR.APPLICATION DATA: APPLICATION NUMBER: US 08/276,852 FILING DATE: 18-JUL-1994,.

INFORMATION FOR SEQ ID NO:l: SEQUENCE CHARACTERISTICS: LENGTH: 173 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO .ZA~ 183 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGCCGCAAAT TCTATTTCAA GGAGACACTC ATAATGAAAT ACCTATTGCC TACGGCAGCC GCTGGATTGT TATTACTCGC TGCCCAACCA GCCATGGCCC AGGTGAAACT GCTCGAGATT 120 TCTAGACTAG TTACCCGTAC GACGTTCCGG ACTACGGTTC TTAATAGAAT TCG 173 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 173 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: TCGACGAATT CTATTAAGAA CCGTAGTCCG GAACGTCGTA CGGGTAACTA GTCTAGAAAT CTCGAGCAGT.TTCACCTGGG CCATGGCTGG TTGGGCAGCG AGTAATAACA ATCCAGCGGC 120 'i TGCCGTAGGC.AATAGGTATT TCATTATGAC TGTCTCCTTG AAATAGAATT TGC 173 i INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 131 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO 184 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TGAATTCTAA ACTAGTCGCC AAGGAGACAG TCATAATGAA ATACCTATTG CCTACGGCAG 60 CCGCTGGATT GTTATTACTC GCTGCCCAAC CAGCCATGGC CGAGCTCGTC AGTTCTAGAG 120 TTAAGCGGCC G 131 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 139 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: *'TCGACGGCCG CTTAACTCTA GAACTGACGA GCTCGGCCAT GGCTGGTTGG GCAGCGAGTA ATAACAATCC AGCGGCTGCC GTAGGCAATA GGTATTTCAT TATGACTGTC TCCTTGGCGA 120 CTAGTTTAGA ATTCAAGCT 139 139 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear i (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 1 5 INFORMATION FOR SEQ ID NO:6: 185 SEQUENCE CHARACTERISTICS: LENGTH: 26 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala S. 1 5 10 Ala Gin Pro Ala Met Ala Gin Val Lys Leu INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 23 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 Ala Gin Pro Ala Met Ala Glu INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 198 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO I r- 186 (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGTTGACAAT TAATCATCGG CTCGTATAAT GTGTGGAATT GTGACCGGAT AACAATTTCA 60 CACAGGAGGA AGGATCCATG AAATACCTAT TGCCTACGGC AGCCGCTGGA TTGTTATTAC 120 TCGCTGCCCA ACCAGCCATG GCCGAGCTCG GTCGGTCGGT CCTCGAGGGT CGGTCGGTCT 180 S. CTAGAGTTAA GCGGCCGC 198 INFORMATION FOR SEQ ID NO:9: w SEQUENCE CHARACTERISTICS: LENGTH: 198 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) *4 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO i: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GCGGCCGCTT AACTCTAGAG ACCGACCGAC CCTCGAGGAC CGACCGACCG AGCTCGGCCA TGGCTGGTTG GGCAGCGAGT AATAACAATC CAGCGGCTGC CGTAGGCAAT AGGTATTTCA 120 TGGATCCTTC CTCCTGTGTG AAATTGTTAT CCGCTCACAA TTCCACACAT TATACGAGCC 180 GATGATTAAT TGTCAACA 198 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: N-terminal 187 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Lys Thr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 Ala Gln Pro Ala Met Ala Glu Leu INFORMATION FOR SEQ ID NO:ll: SEQUENCE CHARACTERISTICS: LENGTH: 220 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular.

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TGTTGACAAT TAATCATCGG CTCGTATAAT GTGTGGAATT GTGACCGGAT AACAATTTCA CACAGGAGGA AGGATCCATG AAATACCTAT TGCCTACGGC AGCCGCTGGA TTGTTATTAC 120 TCGCTGCCCA ACCAGCCATG GCCCAGGTGA AACTGCTCGA GGGTCGGTCG GTCTCTAGAC 180 GCTCGGTCGG TCACTAGTCA TCATCATCAT CATTAAGCTA 220 INFORMATION FOR SEQ ID NO:12 SEQUENCE CHARACTERISTICS: LENGTH: 220 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: "i a-sr- 188 TAGCTTAATG ATGATGATGA TGACTAGTGA CCGACCGACC GTCTAGAGAC CGACCGACCC TCGAGCAGTT TCACCTGGGC CATGGCTGGT TGGGCAGCGA GTAATAACAA TCCAGCGGCT 120 GCCGTAGGCA ATAGGTATTT CATGGATCCT TCCTCCTGTG TGAAATTGTT ATCCGCTCAC 180-" AATTCCACAC ATTATACGAG CCGATGATTA ATTGTCAACA 220 INFORMATION FOR SEQ ID NO:13: SEQUENCE

CHARACTERISTICS:

LENGTH: 28 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide S. FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Lys Thr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 Ala Gln Pro Ala Met Ala Gin Val Lys Leu Leu Glu INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 7 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: C-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Thr Ser His His His His His 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid l -189 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GGCCGCAAAT TCTATTTCAA GGAGACAGTC AT 32 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO o* (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: AATGAAATAC CTATTGCCTA CGGCAGCCGC TGGATT 36 S• INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: '-z>Sv1 190 GTTATTACTC GCTGCCCAAC CAGCCATGGC CC 32 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CAGTTTCACC TGGGCCATGG CTGCTTGGG 29 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic-acid STRANDEDNESS: singlei'': TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)i:; (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: CAGCGAGTAA TAACAATCCA GCGGCTGCCG TAGGCAATAG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) A t~AAA. A~A~,A ~AAA~' A

-SAA~:.

-191 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GTATTTCATT ATGACTGTCT CCTTGAAATA GAATTTGC 38 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: AGGTGAAACT. GCTCGAGATT TCTAGACTAG TTACCCGTAC INFORMATION FOR SEQ ID NO:22:

SEQUENCE.CHARACTERISTICS:

LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CGGAACGTCG. TACGGGTAAC TAGTCTAGAA ATCTCGAG 38 INFORMATION FOR SEQ ID NO:23: w i I ~i 192 SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:.NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: SGACGTTCCGG ACTACGGTTC TTAATACAAT TCG 33 INFORMATION FOR SEQ ID NO:24: 99..

SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 9* (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: TCGACGAATT CTATTAAGAA CCGTAGTC 28 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

-u 11--1- ;-a 193 (xi) SEQUENCE DESCRIPTION: SEQ ID TGAATTCTAA ACTAGTCGCC AAGGAGACAG TCAT 34 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: AATGAAATAC CTATTGCCTA CGGCAGCCGC TGGATT 36 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: .NO :i (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GTTATTACTC GCTGCCCAAC CAGCCATGGC C 31 INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 194 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GAGCTCCTCA GTTCTAGAGT TAACCGGCCG INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single *g TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GTATTTCATT ATGACTGTCT CCTTGGCGAC TACTTTAGAA TTCAAGCT 48 5 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION:. SEQ ID CAGCGAGTAA TAACAATCCA GCGGCTGCCG TAGGCCCAATAG 195 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO S(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TGACGAGCTC GGCCATGGCT GGTTGGG 27 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: TCGACGGCCG CTTAACTCTA GAAC 24..

INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 666 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 196 (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33 O 0 00 *S0 000 0 00 so*** se* 00 *0e 0 CCATTCGTTT GTGAATATCA

AGGCCAAGGC

AATGCTGGCG GCGGCTCTGG

TGGTGGTTCT

GGTGGCGGTT CTGAGGGTGG

CGGCTCTGAG

GGTGATTTTG ATTATGAAA

GATGGCAAAC

GATGAAAACG CGCTACAGTC

TGACGCTAAA

GGTGCTGCTA TCGATGGTTT

CATTGGTGAC

ACTGGTGATT TTGCTGGCTC

TAATTGCCAA

CCTTTAATGA ATAATTTCCG

TCAATATTTA

CCTTTTGTCT TTAGCGCTGG

TAAACCATAT

TTATTCGGTG TCTTTGCGTT

TCTTTTATAT

TTTGCTAACA TAGTGCGTAA

TAAGGAGTCT

TATTAT

INFORMATION FOR SEQ ID NO:34: SEQUENCE

CHARACTERISTICS:

LENGTH: 211 amino ac TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein fiii) HYPOTHETICAL:

NO

FRAGMENT TYPE: internal

GGAGGCGGTT

GCTAATAAGG

GGCAAACTTG

GTTTCCGGCC

ATGGCTCAAG

CGTTCCCTCC

GAATTTTCTA

GTTGCCACCT

TAATCATGCC

CCGGTGGTG

GGGCTATGAC

ATTCTGTCGC

TTGCTAATGG

TCGGTGACGG

CTCAATCGGT

TTGATTGTGA

TTATGTATGT

AGTTCTTTTG

CTGTGGTTCC

CGAAAATGGC

TACTGATTAC

TAATGGTGCT

TGATAATTCA

TGAATGTCGC

CAAAATAAAC

ATTTTCTACG

GGTATTCCGT

CAATCGTCTG ACCTGCCTGA

AGCTCGTGTC

GGTGGCGGCT CTGAGGGTGG TGGCTCTGAG 120 180 240 300 .360 420 480 540 600 660 666 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Pro Phe Val Gys Glu Tyr.Gin Gly Gin Gly Gin Ser Ser Asp Leu Pro 1 5 10 Gin Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly 25 197 Gly Ser Glu Gly Gly Gly Ser Glu Gly Cly Gly Ser Glu Gly Gly Gly 40 Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp 55 Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala 70 75 Asp Glu Asn Ala Leu Gin Ser Asp Ala Lys Gly Lys Leu Asp Ser Val 90 Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly.Asp Val Ser S100 105 110 Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn 115 120 125 Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn 130 135 140 Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gin Ser Val Glu Cys Arg 145 150 155 160 Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys 165 170 175 .Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val 180 185 190 Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn 195 200 205 Lys Glu Ser 210 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 4-:I 198- (xi) SEQUENCE DESCRIPTION: SEQ ID GAGACGACTA GTGGTGGCGG TGGCTCTCCA TTCGTTTGTG AATATCAA 48 INFORMATION FOR SEQ ID NO:36: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: TTACTAGCTA GCATAATAAC GGAATACCCA AAAGAACTGG INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ii: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: TATGCTAGCT AGTAACACGA CAGGTTTCCC GACTGG 36 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 41 -199 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ACCGAGCTCG AATTCGTAAT CATGGTC 27 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: AGCTGTTGAA TTCGTGAAAT TGTTATCCGC T 31 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 708 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID GAGACGACTA GTGGTGGCGG TGGCTCTCCA TTCGTTTGTG AATATCAAGG CCAAGGCCAA n r ~1 200 TCCTCTGACC TCCCTCAACC TCCTGTCAAT GCTGCGCC CCTCTCCTGG

TCCTTCTCGT

GGCCGCTCTG AGGCTCCTCG CTCTGAGGCT GCGCTTCTG ACCCTGGCCG

CTCTGAGGGA

GGCCCTTCCG GTGCTCCCTC TCCTTCCCGGT CATTTTCATT ATCAAAAGAT

GCCAAACCCT

AATAAGGGCC CTATCACCGA AAATGCCGAT CAAAACGCCc TACACTCTCA

CCCTAAAGCC

AAACTTGATT CTGTCGCTAd TCATTACCCT CCTGCTATCG ATGGTTTCAT

TGCTCACGTT

TCCCGCCTTC CTAATCCTAA TGCTOCTACT CCTCATTTTC CTGCCTCTAA

TTCCCMAATG

GCTCAAGTCG GTCACCCTGA TAATTCACCT TTAATGAATA ATTTCCGTCA

ATATTTACCT

TCCCTCCCTC AATCGGTTGA ATCTCGCCCT TTTCTCTTTA GCCCTGGTA ACCATATcAA TTTTCTATTG ATTGTGCAA AATAAACTTA TTCCCTCCTC TCTTTGCGTT

TGTTTTATAT

GTTCCCACCT TTATGTATGT ATTTTCTACG TTTGCTAACA TACTCCGTAA

TAACCAGTCT

TAATCATCCC AGTTCTTTTC GGTATTCCCT TATTATGCTA

GCTAGTA

INFORMATION FOR SEQ ID NO:41: SEQUENCE

CHARACTERISTICS:

LENGTH:. 201 base pairs TYPE: nucleic acid STRANDEDNESS: -double TOPOLOGY: linear (ii)MOLEULETYPE: DNA (genornic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: No SEQUENCE DESCRIPTION: SEQ ID NO:41: TATGCTAGCT *ACTAACACCA CACCTTTCCC CACTCCAAAG CGCCCAGTCA

CCGCAACCCA

ATTAATGTCA GTTAGCTCAC TCATTAGCCA CCCCAGGCTT TACACTTTAT

GCTTCCGCCT

CGTATGTTGT GTGGAATTGT GAGCCATA CAATTTCACA CACGAAACAG

CTATCACCAT

CATTACCAAT TCGAGCTCCC

T

INFORMATION FOR SEQ ID NO:42: SEQUENCE

'CHARACTERISTICS:

LENGTH: 24 base pairs *120 180 240 300 360 420 480 540 600 660 708 120 180 201 S- in>- 201 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: .*CAGGTGCAGC TCGAGCAGTC TGGG 24 INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: .nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii).MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO e (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GAGGTGCAGC TCGAGGAGTC TGGG 24 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO i~ 202 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: GCATGTACTA GTTTTGTCAC AAGATTTGGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO .0 (x i SEQUENCE DESCRIPTION: SEQ ID GACATCGAGC TCACCCAGTC TCCA 24 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear i (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: GAAATTGAGC TCACGCAGTC TCCA 24 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 53 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 203 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO SEQUENCE DESCRIPTION: SEQ ID NO:47: GCGCCGTCTA GAACTAACAC TCTCCCCTGT TGAAGCTCTT TGTGACGGGC AAG 53 S(2) INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 12 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: circular (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: 9 Ser Ile Ser Gly Pro Gly Arg Ala Phe Tyr Thr Gly 1 5 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GTCGTTGACC AGGCAGCCCA

G

SI F S 21 INFORMATION FOR SEQ ID i 204 SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID ATAGAAGTTG TTCAGCAGGC

A

21 INFORMATION FOR SEQ ID NO:51: SEQUENCE

CHARACTERISTICS:

LENGTH: 17 base pairs TYPE: nucleic acid

C

STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: ATTAACCCTC

ACTAAAG

17 INFORMATION FOR SEQ ID NO:52: SEQUENCE

CHARACTERISTICS:

LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

Ack~-. t;,SzAA~0s iS> 205 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: GAATTCTAAA CTAGCTACTT CG 22 INFORMATIONFOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: Leu Glu Glu Ser Gly Thr Glu Phe Lys Pro Pro Gly Ser Ser Val Lys S1 5 10 Val Ser Cys Lys Ala Ser Cly Gly Thr Phe Gly Asp Tyr Ala Ser Asn 25 Tyr Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gln Gly Leu Glu Tyr 40 Ile Gly Gly Ile Thr Pro Thr Ser Gly Ser Ala Asp Tyr Ala Gln Lys 50 55 Phe Gln Gly Arg Val Thr Ile Ser Ala Asp Arg Phe Thr Pro Ile Leu 70 75 e Tyr Met Glu Leu Arg Ser Leu Arg Ile Glu Asp Thr Ala Ile Tyr Tyr 85 90 Cys Ala Arg Glu Arg Arg Glu Arg CGly Trp Asn Pro Arg Ala Leu Arg 100 105 110 Gly Ala Leu Asp Phe Trp Gly Gin Gly Thr Arg Val Phe Val Ser Pro 115 120 125 INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein -206- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: Leu Giu.Giu Ser Cly Ala Ala Val Gin Lys Pro Gly Ser Ser Val Arg 10 Val Ser Cys Gin Ala Ser Gly Gly Thr Phe Asp Asn Phe Ala Ser Asn 25 Tyr Ala Val Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Giu Trp *40 z *Met Gly Gly Ile Thr Pro Thr Ser Gly Thr Ala Thr Tyr-Ser Gin Lys 9Phe Gin Gly Arg Val Thr Ile Ser Ala Ala Pro Leu Thr Pro Ile Ile 70 75 *Tyr Met Giu Leu Arg Ser Leu Arg Asp Asp Asp Thr Aia Val Tyr Tyr 90 Gys Ala Arg Giu Arg Arg Glti Arg Gly Trp Asn Pro Arg Ala Leu Val *Gly Ala Le *u Asp Val Trp Gly Gin Gly Thr Thr Val 115 120 9* INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH:'128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: Leu Giu Glu Ser Gly Thr Giu Phe Lys Pro Pro Gly Ser Ser.ValLy 10 Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Cly Asp Tyr Ala Ser Asn 25 Tyr Ala Ile Ser Trp Vai Arg Gin Ala Pro Gly Gin Gly Leu Giu Tyr 40 Ile Gly Gly Ile Thr Pro Thr Ser Cly Ser Ala Asp Tyr Ala Gin Lys 55 -207- Phe Gin Gly Arg Val Thr Ile Ser Ala Asp Arg Phe Thr Pro Ile Leu 70 75 Tyr Met Giu Leu Arg Ser Leu Arg Ile Giu Asp Thr Ala Ile Tyr Tyr 90 Cys Ala Arg Glu Arg Arg Glu Arg Giy Trp Asn Pro Arg Ala Leu Arg 100 105 110 Gly Ala Leu Asp Phe Trp Gly Gin Gly Thr Arg Val Phe Val Ser Pro 115 120 125 INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid (Dl) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: Leu Glu Giu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys 1 5 10 Val Ser Cys.Lys Ala Ser Gly Gly Ile Phe Ser Asp Phe Ala Ser Asn 25 *Tyr Ala Ile Ser Trp V al Arg Gin Ala Pro Gly Gin Gly Leu Glu Tyr 40 Met Gly Gly Ile Thr Pro Thr Ser Gly Ser Ala Asp Tyr Ala Gin Lys 55 Phe Gin Gly Arg Val Thr Ile Ser Ala Asp Ala Ala Thr Pro Arg Val 70 75 Tyr Met Giu..Leu Arg Ile Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe 85 90 Cys Ala Arg Glu Arg Arg Giu Arg Gly Trp Asn Pro Arg Ala Leu Arg 100. 105 110 Gly Ala Leu Glu Val Trp Gly Gin Gly Thr Thr Val Ile Val Ser Pro 115 .120 125 INFORMATION FOR SEQ ID.NO:57: 208 SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: Leu Glu Glu Ser Gly Ala Ala Val Gin Lys Pro Gly Ser Ser Val Arg 1 5 10 Val Ser Cys Gin Ala Ser Gly Gly Thr Phe Asp Asn Phe Ala Ser Asn 20 25 Tyr Ala Val Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp 35 40 Met Gly Gly Ile Thr-Pro Thr Ser Gly Thr Ala Thr Tyr Ser Gin Lys 55 60 Phe Gin Gly Arg Val Thr lie Ser Ala Ala Pro Leu Thr Pro Ile lle 70 75 Tyr Met Glu Leu Arg Ser Leu Arg Asp Asp Asp Thr Ala Val Tyr Tyr 90 Cys Ala Arg Glu Arg Arg Glu Arg Gly Trp Asn Pro Arg Ala Leu Valii 100 105 110 Gly Ala Leu Asp Val Trp Giy Gin Gly Thr Thr Val Ile Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: Leu Glu Gln.Ser Gly Ala Glu Val Lys Lys Pro Gly Ser Ser Val Lys 1 5 10 -209- Val Ser Gys Lys Thr Ser Gly Gly Thr Phe Ser Asp Tyr Ala Ser Asn 25 His Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Giu Tyr 40* Met Gly Gly Ile Thr Pro Thr Ser Gly Thr Ala Asp Tyr Ala Gin Lys 55 Phe Gin Ala Arg Val Thr Ile Ser Ala His Glu Phe Thr Pro Ile Val 70 75 Tyr Met Giu Leu Arg Ser Leu Arg Ser Asp Gin His Ala Thr Tyr Tyr -85 90 Gys Ala Thr Giu Arg Arg Giu Arg Gly Trp Asn Pro Arg Ala Leu Arg 100 105 110 .Gly Ala Leu Asp Ile Trp Gly Gin Giy Thr Thr Val Ile Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARAGTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESGRIPTION: SEQ ID NO:59: Leu.Glu .Glu Ser Giy Gly Arg Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5. 10 Leu Ser Gys. Glu Gly Ser Gly Phe Thr Phe Thr Asn Ala Trp Met Thr 25- Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile .40 Lys Ser Lys Phe Asp Gly Gly Ser Pro His Tyr Ala Ala Pro Val Glu 55 Gly Arg Phe Ser Ile Ser Arg Asn Asp Leu Giu Asp Lys Met Phe Leu 70 75 Giu Met Ser Gly Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Gys Ala .90 S~ 210 Thr Lys Tyr Pro Arg Tyr Ser Asp Met Val Thr Gly Val Arg Asn His 100 105 110 Phe Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 125 (21) INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Glu Gin Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 L* eu Ser Cys Glu Gly Ser Gly Phe Thr Phe Thr Asn Ala Trp Met Thr 25 *Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 40 Lys Ser Lys Phe Asp Gly Gly Ser Pro His Tyr Ala Ala Pro Val Glu 50 55 Gly Arg Phe Thr Ile Ser Arg Asn Asp Leu Glu Asp Lys Leu Phe Leu 65 70 75 Glu Met Ser Gly Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Cys Ala 90 Thr Lys Tyr Pro Arg Tyr Phe Asp Met Met Ala Gly Val Arg Asn His 100 105 110 Phe TyrMet Asp Val Trp Gly Thr Gly Thr Thr Val Ile Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear s-Ar cc 47 211- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Glu Gly Ser Gly Phe Thr Phe Thr Asn Ala Trp Met Thr 25 Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser lie 35 40 Lys Ser Lys Phe Asp Gly Gly Ser Pro His Tyr Ala Ala Pro Val Glu 50 55 CGly Arg Phe Thr Ile Ser Arg Asn Asp Leu Glu Asp Lys Leu Phe Leu 70 75 Glu Met Ser Gly Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Cys Ala 90 Thr Lys Tyr Pro Arg Tyr Ser Asp Met Met Ala Gly Val Arg Asn His 100 105 110 Leu Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: Leu Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Thr Asn Ser Trp Met Thr 25 Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 40 -SC. +v *~dSC.C-~9 .V 212 Lys Arg Lys Phe Asp Gly Gly Ser Pro His Tyr Ala 55 Gly Arg Phe Ser lie Ser Arg Asn Asp Leu Glu Asp 70 75 .Glu Met Ser Gly Leu Lys Ala Glu Asp Thr GlyVal 90 Thr Lys Tyr Pro Arg Tyr Ser Asp Met Met Thr Gly 100. 105 Phe Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val 115 120 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Ala Lys Tyr Val Ile 125 Pro Val Glu Met Phe Leu Tyr Cys Ala Arg Asn His 110 Val Ser Ser S. S 6e (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: Leu Glu Glu Ser Gly Gly Gly Leu Val Lys 1 5 10 Leu Ser Cys Glu Ser Ser Gly Phe Thr Phe 25 Trp Val Arg Gin Ser Pro Gly Lys Gly Leu 40 Lys Ser Lys Phe Asp Gly Gly Ser Pro His 55 Gly Arg Phe Thr Ile Ser Arg Asn Asp Leu 70 Glu Met Ser Gly Leu Lys Ala Glu Asp Thr 90 Thr Lys Tyr Pro Arg Tyr Ser Asp Met Met 100 105 Phe Tyr Met Asp Val Trp Gly Lys Gly Thr 115 120 Pro Thr Glu Tyr Glu 75 Gly Ala Thr Gly Gly Ser Asn Ala Trp Trp Val Ala Ala Ala Pro Asp Lys Leu Val Tyr Tyr Gly Val Arg 110 Val Ile Val 125 Leu Arg Met Thr Ser Ile Val Glu Phe Leu Cys Ala Asn His Ser Ser xv: ~i~l 213 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid:: TOPOLOGY: linear MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: Leu Glu Glu Ser Gly Gly Arg Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Glu Gly Ser Gly Phe Thr Phe Thr Asn Ala Trp Met Thr 20 25 Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 40 Lys Ser Lys Phe Asp Gly Gly Ser Pro His Tyr Ala Ala Pro Val Glu 55 Gly Arg Phe Ser lie Ser Arg Asn Asp Leu Glu Asp Lys Met Phe Leu 65 70 75 80 Glu Met Ser Gly Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Cys Ala 90 'Thr Lys Tyr Pro Arg Tyr Ser Asp Met Met Thr Gly Val Arg Asn His 100 105 110 Phe Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:65: SEQUENCE .CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 214 Leu Glu Glu Ser Gly Gly Gly Leu Val 1 5 Leu Ser Cys Ala Gly Ser Gly Phe Thr 25 S Trp Val Arg Gin Ser Pro Gly Lys Gly 35 40 Lys Ser Lys Phe Asp Gly Gly Ser Ser Gly Arg Phe Thr lie Ser Arg Asn Tyr 65 70 Glu Met Ser Gly Leu Lys Ala Glu Asp 85 Thr Lys Tyr Pro Arg Tyr Tyr Asp Met 100 105 .Tyr Tyr Met Asp Val Trp Gly Lys Gly 115 120 INFORMATION FOR SEQ ID NO:66: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Gly Ala Val Gly Lys Tyr Val lie 125 Ser Trp Ala Pro Leu Tyr Arg 110 Val Leu Met Ser Val Phe Cys Asn Ser Arg Thr lie Glu Leu Ala His Ser (xi) Leu 1 Val Trp Asn Val SEQUENCE DESCRIPTION: SEQ ID NO:66: Glu Gin Ser Gly Ala Glu Val Lys Lys 5 10 Ser Cys Gin Ala Ser Gly Tyr Arg Phe 25 Val Arg Gin Ala Pro Gly Gin Arg Phe 40 Pro Tyr Asn Gly Asn Lys Glu Phe Ser 55 Thr Phe Thr Ala Asp Thr Ser Ala Asn 70 Pro Gly Ala Ser Val Ser Asn Phe Val Ile Glu Trp Met Gly Trp Ala Lys Phe Gin Asp Thr Ala Tyr Met Glu Vt -215 Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val 90 Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gin Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val lie Val Ser Ser 115 120 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 Val Ser Cys Gin Ala Ser Gly Tyr Arg Phe Ser Asn Phe Val lie His 25 Trp Val Arg Gin Ala Pro Gly Gin Arg Phe Glu Trp Met Gly Trp lie 40 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gin Asp Arg 50 55 Val Thr Phe Thr Ala Asp Thr Asp Ala Asn Thr Ala Tyr Met Glu Leu 65 70 75 Arg Ser Leu Arg Ser Ala Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Val 90 Gly Pro Tyr Thr Trp Asp Asp Ser Pro Gin Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Lys Val Ile Val Ser Ser 115 120 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear zzc-a~~4 v4JSSA$±xv 216 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys 1 5 10 Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Phe Val Ile His 25 Trp Val Arg Gin Ala Pro Gly Gin Arg Phe Glu Trp Met Gly .Trp Ile 35 40 Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe Gin Asp Arg 50 55 Val Thr Phe Thr Ala Asp Thr Asp Ala Asn Thr Ala Tyr Met Glu Leu 70 75 Arg Ser Leu Arg Ser Thr Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Val 85 90 Gly Pro Tyr Thr Trp Asp Asp Ser Pro Gin Asp Asn Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Lys Val Ile Val Ser Ser 115 120 2) INFORMATION FOR SEQ ID NO:69: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 130 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Ser Ser Ala Trp Met Ala 25 Trp Val Arg Gin Ala Pro Gly Arg Gly Leu Glu Trp Val Gly Leu Ile 40 -217- Lys Ser Lys Ala Asp Gly Glu Thr Thr Asp Tyr Ala 55 Gly Arg Phe Ser Ile Ser Arg Asn Asn Leu Glii Asp 70 75 *Gin Met Asp Ser Leu Arg Ala Asp Asp Thr Ala Val 90 *Thr Gin Lys Pro Arg Tyr Phe Asp Leu Leu Ser Gly 100 105 Val Ala Gly Ala Phe Asp Val Trp Gly His Gly Thr 115 120 Ser Pro 130 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 130 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULETYPE: protein Thr P Thr V Tyr TI Gin T1 1 Thr N 125 Gly Ala Val Thr Thr 1Tyr y Gin ro Val Lys 'al Tyr Leu ~yr Cys Ala 'yr Arg Arg lal Thr Val (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Glu Clii Ser Gly Gly Gly L eu Val Lys Ala 1 5 10 Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Ser 20 *25 Trp Val Gly Gin Ala Pro Gly Arg Gly Leu Clii 40 Lys Ser Lys Ala Asp Gly Glu Thr Thr Asp Tyr *55 Gly Arg Phe Ser Ie Ser Arg Asn Asn Leu Clii 70 75 Gin Met Asp Ser Leu Arg Ala Asp Asp*Thr Ala 90 Thr Gin Lys Pro Arg Tyr Phe Asp Leu Leu Ser 100 105 C ly Ser Trp Ala Asi Va~ Cl' Ser Leu Arg Trp Met Ala Gly Leu Ile Pro Val Lys Val Tyr Leu Tyr Cys Ala Tyr Arg Arg 110 218 Val Ala Gly Ala Phe Asp Val Trp Gly His Gly Thr Thr Val Thr Val 115 120 125 Ser Pro 130 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: LENGTH: 130 amino acids TYPE: amino acid TOPOLOGY: linear 4* (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: Leu Glu Glu Ser Gly Gly Gly Leu Ile Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Val Gly Ser Gly Phe Thr Phe Ser Ser Ala Trp Met Thr 20 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Leu Ile 35 40 Lys Ser Lys Ala Asp Gly Glu Thr Thr Asp Tyr Ala Thr Pro Val Lys 55 Gly Arg Phe Thr Ile Ser Arg Asn Asn Leu Glu Asn Thr Val Tyr Leu 65 70 75 Gin Met Asp Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala 90 Thr Gin Lys Pro Ser Tyr Tyr Asn Leu Leu Ser Gly Gin Tyr Arg Arg 100 105 110 Val Ala Gly Ala Phe Asp Val Trp Gly His Gly Thr Thr Val Thr Val 115 120 125 Ser Pro INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear j;~llll~~ 219 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: Leu Glu Glu Ser Gly Glu Ala Val Val Gin Pro Gly Arg Ser Leu.Arg 1 5 10 Leu Ser Cys Ala Ala Ser Gly Phe lie Phe Arg Asn Tyr Ala Met His 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu lie S. 35 40 Lys Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg 50 55 Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gin Met 70 75 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp 90 lie Gly Leu Lys Gly Glu His Tyr Asp Ile Leu Thr Ala Tyr Gly Pro 100 105 110 Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS:: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: Leu Glu Gin Ser Gly Glu Ala Val Val Gin Pro Gly Thr Ser Leu Arg 1 5 10 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr Ala Met His 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile 40 I;iiiii~i:i~ 220 Lys Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser 55 Phe Ser lle Ser Arg Asp Asn Ser Lys Asn Thr Leu 70 75 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 90 lie Gly Leu Lys Gly Glu His Tyr Asp Ile Leu Thr 100 105 Asp Tyr Trp Gly Gin Gly Ala Leu Val Thr Val Ser 115 120 INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein.

S**

6* 00* 0

S

0 °oooo° oooo Val Tyr Cys Ala Ser 125 Arg Tyr Val Val Tyr Cys Ala Ser 125 Lys Gly Arg Leu Glu Met Ala Arg Asp Tyr Gly Pro 110 (xi) Leu 1 Leu Trp Lys Phe Asn lie Asp SEQUENCE DESCRIPTION: SEQ ID NO:74: Glu Gin Ser Gly Glu Ala Val Val Gin 5 Ser Cys Ala Ala Ser Gly Phe lie Phe 20 Val Arg Gin Ala Pro Gly Lys Gly Leu 40 Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala 55 Thr Ile Ser Arg Asp Asn Ser Lys Asn 70 Ser Leu Arg Ala Glu Asp Thr Ala Val Gly Leu Lys Gly Glu His Tyr Asp Ile 100 105 Tyr Trp Gly Gin Gly Thr Leu Val Thr 115 120 Gly Asn Trp Ser Leu Tyr Thr Ser Arg His Ile Arg Met Asp Pro S- A±2ACZ. S -221 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein r (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Glu Glu Ser Gly Glu Ala Val Val Gin Pro Gly Thr Ser Leu Arg 1 5 10 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr Ala Met His 20 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile 40 Lys Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala Asp Ser Val Lys Gly Arg 55 Phe Ser Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Glu Met 65 70 75 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp 90 Ile Gly Leu Lys Gly Glu His Tyr Asp Ile Leu Thr Ala Tyr Gly Pro 100 105 110 Asp Tyr Trp Gly Gin Gly Ala Leu Val Thr Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: Leu Glu Gln Ser Gly Glu Ala Val Val Gln Pro Gly Arg Ser Leu Arg 1 5 10

I~

0.

*o e oo oooeo 222 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 40 Lys Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala 55 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 65 70 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 90 lie Gly Leu Lys Ala Glu His Tyr Asp lie 100 105 Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr 115 120 INFORMATION FOR SEQ ID NO:77: SEQUENCE

CHARACTERISTICS:

LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Arg Glu Asp Thr 75 Tyr Leu Val Pro Arg I Glu T isp S 6 hr L 5 yr T Asn Tyr Ala Trp Val Ala Ser Val Lys Leu Tyr Leu Tyr Cys Ala Thr Ala Tyr 110 Ser Ser 125 Me Le Gl Glr Arg Gly t His u Ile y Arg n Met Asp Pro Arg His Ile Arg Met Asp (xi) Leu 1 Leu Trp Lys Phe Asn SEQUENCE DESCRIPTION: SEQ ID NO:77: Glu Gin Ser Gly Glu Ala Val Val Gin 5 10 Ser Cys Ala Ala Ser Gly Phe Ile Phe 25 Val Arg Gln Ala Pro Gly Lys Gly Leu 40 Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala 55 Thr Ile Ser Arg Asp Asn Ser Lys Asn 70 Ser Leu Arg Ala Glu Asp Thr Ala Val 90

C

T

7

T

Sly Arg Asn Tyr rp Val er Val 0 ,eu Tyr yr Cys Ser Leu Ala Met Ala Leu Lys Gly Leu Gin Ala Arg i-n 223 lie Gly Leu Lys Gly Glu His Tyr Asp lie Leu Thr Ala Tyr Gly Pro 100 105 110 Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: Leu Glu Gin Ser Gly Gly Gly Val Val Lys Pro Gly Gly Ser LeuArg 1 5 10 Leu Ser Cys Glu Gly Ser Gly Phe Thr Phe Pro Asn Ala Trp Met Thr 25 Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile 35 40 Lys Ser Lys Phe Asp Gly Gly Ser Pro His Tyr Ala Ala Pro Val Glu 55 Gly Arg Phe Thr Ile Ser Arg Asn Asp Leu Glu Asp Lys Val Phe Leu 70 75 Gin Met Asn Gly Leu Lys Ala Glu Asp Thr Gly Val Tyr Tyr Cys Ala 90 Thr Arg Tyr Pro Arg Tyr Ser Glu Met Met Gly Gly Val Arg Lys His 100 105 110 Phe Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ser Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Th.- -224 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: Leu Giu Giu Ser Gly Gly Gly Vai Val Lys Pro Oly Gly Ser Leu Arg 10 Leu Ser Cys Giu Gly Ser Gly Phe Thr Phe Pro Asn Ala Trp Met Thr .25 Trp Val Arg Gin Ser Pro Gly Lys Gly Leu Glu Trp Val Ala Ser Ile *35 .40. Lys Ser Lys Phe Asp Gly Gly Ser Pro His Tyr Ala Ala Pro Val Giu 55 Gly Arg Phe Thr Ile Ser Arg Asn Asp Leu Glu Asp Lys Val Phe Leu *65 70 75 Gin Met Asn Gly Leu Lys Ala Giu Asp Thr Giy Val Tyr Tyr Cys Ala 90 Thr Arg Tyr Pro Arg Tyr Ser Giu Met Met Gly Gly Val Arg Lys His 100 105 110 *Phe Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ser Val Ser Ser 115 120 125 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 122 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: -protein (xi) SEQUENCEIDESCRIPTION: SEQ ID Leu Giu Glu Ser Gly Giy Gly Leu Val Gin Pro Gly Arg Ser Leu Arg 1 5 10i Vai Ser Cys .Giu Ala Ser Gly Phe Thr Phe Ser Ser Tyr Giu Met Asn 25 Trp Vai Arg Gin Aia Pro Giy Lys Gly Leu Glu Trp Val Ser Gin Ile 40 Ser Ser Ser Gly Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg >.V$I;.,sssw- _Zvi~-.4f+~m 225 55 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 70 Thr Ser Leu Arg Vai Asp Asp Thr Ala Val 90 _Arg Arg Leu Vai Thr Phe Gly Gly Vai Val 100 .0 Gly Gin Gly Thr Met Val Thr Val Ser Ser 115 120 INFORMATION FOR SEQ ID NO:8i: SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLEC .ULE TYPE.: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: Leu Giu Gin Ser Gly Gly Giy Vai Val Gin 1 5 10 Leu Ser 'Cys Ala Gly -Ser Giy Phe Asn Phe Trp Val .Arg Gin Ala Pro Gly Lys Gi y Leu 35 Ser Tyr Glu Gly Ser Asp Lys Tyr Tyr Ala Phe Thr Ile Ser Arg Asp Asn Ser Giu Asn Asp Ser Leu Arg Ala Asp Asp Thr Ala Leu 90 Thr Arg Giu Asn Ile Giu Ala Asp Gly Thr 100 105 Met Asp Val Trp Gly Lys Gly ThrThr Val 115 120 INFORM4ATION FOR SEQ ID NO:82: Ser Leu Tyr Leu Giu Met 75 Tyr Tyr Gys Ala Arg Giy Ser Gly Gly Asn Ile Trp 110 Is.

Leu Met Val C ly Gin Arg Tyr -226 SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 Val Thr Ile Thr Cys Arg Ala Ser Gin Gly lie Ser Asn Tyr Leu Ala 20 25 Trp Tyr Gin Gin Lys Pro Gly Lys Val Pro Arg Leu Leu lie Tyr Ala 35 40 Ala Ser Thr Leu Gin Pro Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 55 Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp 65 70 75 80 Val Ala Thr Tyr Tyr Cys Gin Lys Tyr Asn Ser Ala Pro Arg Thr Phe 90 Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Ile Gly Asp Arg 1 5 10 Val Thr Ile Thr Cys Arg Ala Ser Gin Gly Ile Asn Asn Tyr Leu Ala 25 Trp Tyr Gin Gin Arg Pro Gly Lys Val Pro Arg Leu Leu Ile Tyr Ala 227 9* 40 Ala Ser Thr Leu Gin Ser Gly Val Pro Thr 55 Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 65 70 SVal Ala Thr Tyr Tyr Cys Gin Lys Tyr Asn 85 90 Gly Gly Gly Thr Lys Val Glu lie Lys Arg 100 105 INFORMATION FOR SEQ ID NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid (D).TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser 1 5 Val Thr lie. Thr Cys Arg Ala Ser Gin Gly 25 Trp Tyr Gin Gin Lys Pro Gly Lys Val Pro 40 Ala.Ser Thr Leu Gin Ser Gly Val Pro Ser .55 Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 70 Val Ala Thr Tyr Tyr Cys Gin Lys Tyr Asn Gly Gin Gly Thr Lys Val Glu Ile Lys Arg 100 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acids Arg Phe Ser Gly Ser Gly Ser Leu Gin Pro Glu Asp 75 Ser Val Pro Arg Thr Phe Ala Ile Lys Arg Ser Ser Thr Ser Ser Leu Phe 60 Leu Ala Val Asn Leu Ser Gin Pro Gly Tyr 30 Ile Gly Pro Arg i: I i j: i F;I:liri C ~I c~ 228 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 253 Trp yr Gl h Gin rg Pro Ser LsLur Ala sner Le Ile Asp Arg 35 5415 1 Val Thr lie Thr Cys Ar la Ser Ine Gy le Asn Asn Tro lu Ala 25 30 8 Trp Tyr Gin Gin Ar Pro Gly Lys Alayros Asn Vlu Peo Hie Tyr Ala 5 40 Ala SerGl Thr LeuGnsey Val Pro Pro Arg PhAerCySe l 100 0 (Ar LENTy Thr Amip Phahceuirdes e e LuGnPr l s Vl la Thr Tyr Tyr Cys GiLys Tyr A Ser ValSe Pro His Th r Ph 1 5 90 Glyal l Thr L rCys rAlSeGnSr Val Gle lie Lys ArgLe INOMTION Or SEQ IDNOsPo:86: nAaPr r LuL l-y 35 SEUEC CHRCERSIS Gly Val Ser Asn Arg Ala Thr 55 Gly Ser Gly Thr Asp Phe Thr 70 Asp Phe Ala Val Tyr Ser Cys Phe Gly Gin Gly Thr Lys Val 100 INFORMATION FOR SEQ ID NO:87 SEQUENCE CHARACTERISTIC: LENGTH: 107 amino TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 229 Gly Ile Pro Asp Arg Phe Ser Gly Ser Leu Thr Ile Ser Arg Leu Glu Pro Glu 75 Gin Gin Tyr Gly Thr Ser Pro Trp Thr 90 Glu Ile Lys Arg Thr 105 eo ooo oooo eee ee (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser 1 5 10 Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser 20 25 Ala Trp Tyr Gin Gin Arg Pro Gly Gin Ala 40 Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro 55 Gly Ser Gly Thr Ala Phe Thr Leu Thr Ile 70 Asp Val Ala Ile Tyr Tyr Cys Gln Gin Tyr 90 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys 100 105 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear Leu Val Pro Asp Ser 75 His Arg Ser Ser Arg Arg Ser Ser Pro Asn Leu Phe Leu Ser Gly Asn 30 Leu Ser Gin Pro Glu Arg 15 Tyr Leu Ile Tyr Gly Ser Pro Glu 80 Tyr Thr i I ~i.

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230 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 Ala Thr Leu Ser Cys Arg Ala Ser His Arg Val Asn Asn Asn Phe Leu 20 25 Ala Trp Tyr Gin Gin Lys Pro Gin Ala Pro Arg Leu Leu Ile Ser Gly 35 40 Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly 55 Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Arg Leu Glu Pro Asp Asp 70 75 Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Asp Ser Pro Leu Tyr Ser 85 90 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Thr 100 .105 INFORMATION FOR SEQ ID NO:89: SEQUENCE CHARACTERISTICS: LENGTH: 105 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: Glu Leu Thr Gin Ser Pro Ala Ser Val Ser Ala Ser Val Gly Asp Thr 1 5 10 Val Thr Ile Thr Cys Arg Ala Ser Gin Asp Ile His Asn Trp Leu Ala 25 Trp Tyr Gin Gin Gin Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 40 Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Arg Gly 55 231 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp 70 75 Phe Ala Thr Tyr Tyr Cys Gin Gin Gly Asn Ser Phe Pro Lys Phe Gly 90 Pro Gly Thr Val Val Asp Ile Lys Arg 100 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 5 10 Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Leu Ser Asn Asn Tyr Leu 20 25 Ala Trp Tyr Gin Gin Lys Pro Gly Gln Ala Pro Arg Leu Leu lie Tyr 40 Gly Ser Ser Thr Arg Gly Thr Gly Ile Pro Asp Arg Phe Ser Gly Gly.-..

50 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu ii' 70 75 Asp Phe Ala Val Tyr Tyr Cys Gin His Tyr. Gly Asn Ser Val Tyr Thr 90 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg 100 105 INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein o o -232 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: Gin Ser Pro Asp Thr Leu Ser Leu Asn Pro 1 5 10 .Ser Cys Arg Ala Ser His Arg lie Ser Ser 25- Gin His Lys Arg Gly Gin Ala Pro Arg Leu 35 40 Asn Arg Ala Gly Gly Val Pro Asp Arg Phe 50 Thr Asp Phe Thr Leu Thr Tyr Ser Arg Leu 70 Met Tyr Tyr Cys Gin Tyr Tyr Gly Gly Ser 85 90 Gly Thr Lys Val Glu Ile Thr Arg 100 INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: Gin Ser Pro Ser His Leu Ser Leu Ser Pro 1 5 Ser Cys Arg Ala Ser Gin Arg Val Ser Ala 25 Gin Gin Arg Pro Gly Gin Ala Pro Arg Leu 40 Thr Arg Ala Thr Asp Ile Pro Asp Arg Phe 55 Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 70 Arg Ala Leu Ala Tyr Val Ser Gly Glu Asp Thr Phe Thr Trp Cys Ser Phe Gly Leu Tyr Pro Gly Ala Gin :41 :i: Leu Tyr Ser Gly Ala Gly Glu Arg Ala Pro Tyr Leu Ala Val lie Tyr Gly Ser Gly Ser Gly Glu Pro Glu Asp 75 7-v-+rs'+c i 233 lie Tyr Tyr Cys Gin Val Tyr Gly Gin Ser Pro Val Leu Phe Gly Gin 90 Gly Thr Lys Leu Glu Met Lys Arg i 100 INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 105 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Asp Arg Ala Thr Leu 1 5 10 15 Ser Cys Arg Ala Ser Gin Ser Leu Ser Ser Ser Phe Leu Ala Trp Tyr 20 25 Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr Ser Ala Ser 35 40 Met Arg Ala Thr Gly Ile Pro Asp Arg Phe Arg Gly Ser Val Ser Gly 55 60 i i Thr Asp Phe Thr Leu Thr. lie Thr Arg Leu Glu Pro Glu Asp Phe Ala 65 70 75 Val Tyr Tyr Cys Gin Arg Phe Gly Thr Ser Pro Leu Tyr Thr Phe Gly 90 95 i Gin Gly Thr Lys Leu Glu Met Lys Arg 100 105 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: 234 Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu 1 5 10 Ser Cys Arg Ala Ser Gin Ser Phe Ser Ser Asn Phe Leu Ala Trp Tyr 25 Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie Tyr Val His Pro 40 Asn Arg Ala Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly 50 55 S: Thr Asp Phe Thr Leu Thr Ile Arg Arg Leu Glu Pro Glu Asp Phe Ala 65 70 75 Val Tyr Tyr Cys Gin Gin Tyr Gly Ala Ser Leu Val Ser Phe Gly Pro 85 90 Gly Thr Lys Val.His Ile Lys Arg 100 INFORMATION FOR SEQ ID NO:95: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein i (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu.Ser Pro Gly Glu Arg 1 5 10 Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile Arg Ser Arg Arg Val 25 Ala Trp Tyr Gin His Lys Pro Gly Gin Ala Pro Arg Leu Val Ile His 40 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser Gly .Ser 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu Pro Glu 70 75 Asp Phe Ala .Leu Tyr Tyr Cys Gin Val Tyr Gly Ala Ser Ser Tyr Thr 90 235 Phe Gly Gin Gly Thr Lys Leu Clu Arg Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:96: SEQUENCE

CHARACTERISTICS:

LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Thr Pro Gly Glu Arg 1 5 10 Ala Thr Leu Ser Cys Arg Thr Ser His Ser Ile Arg Ser Arg Arg Leu 25 30 Ala Trp Tyr Gin Val Lys Gly Gly Gin Ala Pro Arg Leu Leu Ile Tyr 35 40 Gly Val Ser Asn Arg Ala Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu 70 75 Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Ser Arg Tyr Thr 85 90 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:97: i SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: Glu Leu Thr Gin Ala Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 236 Ala Thr Phe Ser Cys Arg Ser Ser His 25 Arg Trp Tyr Gin His Lys Pro Gly Gln 40 SGly Val Ser Asn Arg Ala Ser Gly Ile 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr 70 Asp Phe Ala Leu Tyr Tyr Cys Gin Val Phe Gly Gin Gly Thr Lys Leu Glu Arg 100 105 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Ser Ala Ser lle Tyr 90 Lys Ile Arg Ser Arg Pro Arg Leu Val Asp Arg Phe Ser Thr Arg Val Glu 75 Gly Ala Ser Ser Arg Arg Val Ile His Gly Ser Pro Glu Tyr Thr Asp Arg Arg Val Ile His Gly Ser Pro Glu Tyr Thr (xi) Glu 1 Ala Ala Gly Gly Asp Phe SEQUENCE DESCRIPTION: SEQ ID NO:98: Leu Thr Gln Ala Pro Gly Thr Leu Ser 5 10 Thr Phe Ser Cys Arg Ser Ser His Asn .25 Trp Tyr Gln His Lys Pro Gly Gln Ala 40 Val Ser Asn Arg Ala Ser Gly Ile Ser 55 Ser Gly Thr Asp Phe Thr Leu Thr Ile 70 Phe Ala Leu Tyr Tyr Cys Gin Val Tyr 90 Gly Gin Gly Thr Lys Leu Asp Phe Lys 100 105 'i- 'i i; :i i i; i i' :r i Leu Ser Ile Arg Pro Arg Asp Arg Thr Arg 75 Gly Ala Arg Thr Gly Arg 30 Val Ser Glu Ser 237 INFORMATION FOR SEQ ID NO:99: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:99: Giu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg 1 5 10 9** Ala Thr Leu Ser Cys Arg Ala Gly Gin Ser lie Ser Ser Asn Tyr Leu 25 Ala Trp Tyr Gin Gin Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 40 Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser 55 Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Arg Leu Glu Pro Glu 65 70 75 Asp Phe Ala Val Tyr.Tyr Cys Gin Gin Tyr Gly Thr Ser Pro Tyr Thr 90 "Phe Gly Gin Gly Thr Gin Leu Asp Ile Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE:.protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu 1 5 10 Ser Cys Arg Ala Ser Gin Ser Leu Ser Asn Asn Tyr Leu Ala Trp Tyr 25 YZ4~A~4 A*ATh AA4Z A$AA> A *oo ne

S

S

o o o o Gln Gln Lys Pro Gly Gin Ala Thr Arg Ala Thr Gly Ile Pro 55 Thr Asp Phe Thr Leu Thr Ile 70 Val Tyr Tyr Cys Gin Gin Tyr Gly Thr Lys Leu Glu Ile Lys 100 INFORMATION FOR SEQ ID NO:101 SEQUENCE CHARACTERISTICS LENGTH: 106 amino a TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 238 Pro Arg Leu Leu Ile Tyr Gly Ser Ser 40 Asp Arg Phe Ser Gly Gly Gly Ser Gly Ser Arg Leu Glu Pro Glu Asp Phe Ala 75 Gly Asn Ser Val Tyr Thr Phe Gly Gin 90 Arg (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: Glu Leu Thr Gin Ser.Pro Ser Ser Leu Ser 1 5 10 Val Thr Ile Thr Cys Arg Thr Ser Gin Gly Trp Tyr Gin Gin Lys Pro Gly Lys Val Pro 35 Ala Ser Thr Leu Gin Ser Gly Gly Pro Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Val Ala Thr Tyr Ser Cys Gin Asn Tyr Asp 90 Gly Gin Gly Thr Lys Val Asp Ile Lys Arg 100 105 INFORMATION FOR SEQ ID NO:102: SEQUENCE CHARACTERISTICS: Asp 15 Leu Tyr Ser Glu Thr

I

i i -11 i: :1 i ;ii i. *iiiiij 239 LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 **Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile Ser Asn Tyr Leu Asn 25 Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 35 40 .4 **Ala Ser Ser Leu Gin Arg Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 55 Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser Ser Leu Gin Pro Glu Asp 70 75 Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Ile Pro Pro Leu Thr 90 000* 0" oPhe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:103: 0(i) SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE.TYPE: protein (xi) SEQUENCE DESCRIPTION:.SEQ ID NO:103: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 Val Thr Ile Thr Cys Arg Ala Ser Gin Asn Ile Asn Asn Tyr Leu Asn 25 Trp Tyr Gin Gin Lys Pro Gly Giu Ala Pro Lys Leu Leu Ile His Thr 40 Ala Phe Asn Leu Gin Ser Gly 55 Ser Gly Thr Glu Phe Thr Leu 70 Phe Ala Thr Tyr Tyr Cys Gin Gly Gin Gly Thr Lys Val Glu 100 INFORMATION FOR SEQ ID NO:104 SEQUENCE CHARACTERISTICS LENGTH: 107 amino a TYPE: amino.acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 240 Val Pro Ser Arg Phe Ser Gly Thr Ala Thr lie Arg Ser Leu Gin Pro Glu Asp 75 80 Gin Ser Tyr Ser Thr Pro Tyr Thr Phe S 90 Ile Lys Arg Thr 105 il ii'i:; (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser 1 5 Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 70 Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg 100 105 INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear :i l i i ;i$dl'; r. 241 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile Ser Ser Tyr Leu Asn 25 Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 35 40 Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 50 55 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp 65 70 75 Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Thr Pro Gin Thr Phe 85 90 95 Gly Gin Gly Thr Lys Leu Glu lie Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE.: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 1 5 10 Thr Cys Arg Ala Ser Gin Thr lie Ser Ser Tyr. Leu Asn Trp Tyr Gin 25 Cin Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser 40 Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Gly Gly Ser Gly Thr 55 -242 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr 70 75 Tyr Tyr Cys Gin Gin Ser Tyr Ser Thr Pro Tyr Thr Phe Gly Gin Gly 90 Thr Lys Leu Glu Ile Lys Arg Thr 100 INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: '.Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 1 5 10 "Val Thr Ile Thr Cys Gin Ala Ser Gin Asp Ile Arg Asn Tyr Leu Asn 20 25 Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Asp 40 Ala Ser Asn. Ser Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 50 55 Ser Gly Arg Asp Phe Thr Phe Thr Ile Ser Ser Leu Gin Pro Glu Asp 70 75 Val Ala Thr Tyr Tyr Cys Gin Gin His Gin Asn Val Pro Leu Thr Phe 90 Gly Gly Gly Thr Lys Val Glu lie Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:108: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein *o .o 243 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: Clu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala 1 5 10 Val Thr lie Thr Cys Gin Ala Ser Gin Asp lie 25 Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 40 Ala Ser Asn Leu Glu Thr GlyVal Pro Ser Arg 55 Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser 65 70 75 lie Ala Thr Tyr Tyr Cys Gin Gin Tyr Asp Asn 90 Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Ser Val Gly Asp Ser Asn His Leu Leu Leu Ile Tyr Phe Ser Gly Ser Leu Gin Pro Glu Leu Pro Leu Thr Arg Asn Asp Gly Asp Phe :li i: :i i i i i i: (xi) Clu 1 lie Trp Ala Ser SEQUENCE DESCRIPTION: SEQ ID NO:109: Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala 5 10 Thr Ile Thr Cys Arg Ala Ser Gin Thr lie Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys 40 Ser Asn Leu Gin Ser Gly Val Pro Ser Arg 55 Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser 70 75 Ser Val Asn Asn Leu Leu Phe Ser Leu Gin Asp Leu Tyr Ser Glu Arg Asn Gly Gly Asp ;-07 t< 244 'Phe.Ala Thr Tyr Phe Cys Gin Gin Ser Tyr Asn Thr Pro Pro Trp Thr 90 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 (2)-INFORMATION FOR SEQ ID NO:110: SEQUENCE CHARACTERISTICS: LENGTH: 108 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Giu Arg 1 5 10 .Ala Thr Leu Ser Cys Arg Ala Ser Gin Arg Val Asn Ser Asn Tyr Leu 25 Ala Trp Tyr Gin Gin Lys Pro Gly Gin Thr Pro Arg Val Val Ile Tyr 40 Ser Thr Ser ArgArg Ala Thr Gly Val Pro Asp Arg Phe Ser Gly Ser 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu 70 75 Asp Phe Ala Val Tyr Tyr Cys Gin Gin Phe Gly Asp Ala Gin Tyr Thr 90 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Thr 100 105 INFORMATION FOR SEQ ID NO:111: SEQUENCE CHARACTERISTICS: LENGTH: 93 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: -245 Giu Arg Ala Thr Leu Ser Cys Arg Ala 1~ 5 Tyr Leu Ala Trp Tyr Gin Gin Lys Pro 25 Ile Tyr Ser Thr Ser Arg Arg Ala Thr 40 Gly.Ser Gly Ser Gly Thr Asp Phe Thr 55 Pro Giu Asp Phe Ala Val Tyr Tyr Cys 65 70 Tyr Thr Phe Gly Gin Gly Thr Lys Leu 85 INFORMATION FOR SEQ ID NO:112: SEQUENCE

'CHARACTERISTICS:

LENGTH: 104 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Ser Gin 10 Gly Gin Giy Val Leu Thr Gin Gin 75 Ciu Ile Arg Val Asn Ser Thr Pro Arg Val Pro Asp Arg Phe Ile Ser Arg Leu Phe Cly Asp Ala Lys Ar'g (xi) Thr Phe His Asp Thr Thr Thr SEQUENCE DESCRIPTION: SEQ ID NO:i12: Gin Ser Pro Ser Ser Val Ser Ala 'Ser 5 10 Thr Cys Arg Ala Ser Gin Asp Ile Arg 25 Gin Lys Pro Gly Lys Ala Pro Lys Leti 40 Leu Giu le-Gly Val Pro Ser Arg Phe 55 Tyr Phe Ser Phe Thr Ile Ser Ser Leu 70 Tyr Tyr Cys Gin Gin Tyr Ala Asp LeuI 90 Lys Val Giu*le Lys Arg Thr 100 Val Asn Leu Ser 1ln lie C ly Tyr Ile C ly Pro Thr Asp Leu Ser Ser Ciu Phe Thr Val Asn Trp Asp Ala Cly Ser Asp Ile Cly Gly

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8 Asn Val1 Ser Giu Gin IThr C'yr 3er lia ;iy liy 246 INFORMATION FOR SEQ ID NO:113: SEQUENCE

CHARACTERISTICS:

LENGTH: 96 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val 1 5 10 Gly Thr Asn Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg 25 Leu Leu Ile Phe Asp Ala Ser Thr Arg Asp Thr Tyr Ile Pro Asp Thr 40 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ala Leu Thr Ile Ser Ser 55 Leu Gin Ser Glu AsP Phe Gly Phe Tyr Tyr Cys Gin Gin Tyr Asp Asn 65 70 75 Trp Pro Pro Thr Phe Gly Gin Gly Thr Lys Leu Glu Val Lys Arg Thr 90 INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Asp Arg 1 5 10 Ala Thr Phe Ser Cys Arg Ser Ser His Asn Ile Arg Ser Arg Arg Val 25 Ala Trp Tyr Gin His Lys Pro Gly Gin Ala Pro Arg Leu Val Ile His 247 40 Gly Val Ser Asn Arg Ala Ser Gly Ile Ser 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 70 Asp Phe Ala Leu Tyr Tyr Cys Gin Val Tyr 90 Phe Gly-Gn Gly Thr Lys Leu Asp Phe Lys 100 105 INFORMATION FOR SEQ ID NO:115: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser 1 5 10 Ala Thr Phe Ser Cys Arg Ser Ser His Asn Ala Trp Tyr Gin His Lys Pro Gly Gln Ala 40 Gly Val Ser Asn Arg Ala Thr Gly Ile Ser 55 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 70 Asp Phe Ala Leu Tyr Tyr Cys Gin Val Tyr 90 Phe Gly Gin Gly Thr Lys Leu Asp Phe Lys 100 105 INFORMATION FOR SEQ ID NO:116: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids Asp Arg Phe Ser Gly Ser Thr Arg Leu Glu Pro Glu 75 Gly Ala Ser Ser Tyr Thr Arg Leu Ile Pro Asp Thr 75 Gly Arg Ser Arg Arg Arg Arg Ala Pro Ser Leu Phe Leu Ser Glu Arg Ile Gly Pro Tyr Arg Val His Ser Glu Thr 248 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: Glu Leu Thr Gin Ser Pro Asp Thr Leu Ser Leu Asn Val Gly Glu Arg 1 5 10 Ala Thr Leu Ser Cys Arg Ala Ser His Arg Ile Ser Ser Arg Arg Leu 20 25 Ala Trp Tyr Gin His Lys Arg Gly Gin Ala Pro Arg Leu Leu Ile Tyr 35 40 Gly Val Ser Ser Arg Ala Gly Gly Val Pro Asp Arg Phe Ser Gly Ser 55 60 Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Arg Leu Glu Pro Glu 70 75 Asp Phe Ala Met Tyr Tyr Cys Gin Thr Tyr Gly Gly Ser Ser Tyr Thr 90 Phe Gly Gin Gly Thr Lys Val Asp Ile Lys Arg 100 105 INFORMATION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: LENGTH: 107.amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: Glu Leu Thr Gin Ser Pro Asp Thr Leu Ser Leu Asn Ala Gly Glu Arg 1 5 10 Ala Thr Leu Ser Cys Arg Ala Ser His Arg Ile Ser Ser Arg Arg Leu 25 Ala Trp Tyr Gin His Lys Arg Gly Gin Ala Pro Arg Leu Leu lie Tyr 40 Gly Val Ser Asn Arg Ala Gly 55 Gly Ser Gly Thr Asp Phe Ser 70 Asp Phe Ala Ile Tyr Tyr Cys Phe Gly Gln Gly Thr Thr Val 100 INFORMATION FOR SEQ ID NO:118 SEQUENCE CHARACTERISTICS LENGTH: 107 amino a TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 249 Gly Val Pro Asp Arg Phe Ser Gly Ser Leu Thr Ile Ser Arg Leu Glu Pro Glu 75 Gin Thr Tyr Gly Gly Ser Ser Tyr Thr 90 Asp Ile Lys Arg 105 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: Glu Leu Thr Gin Ser Pro Asp Thr Leu Ser Leu 1 5 10 Ala Thr Leu Ser Cys Arg Ala Ser His Arg lie 25 Ala Trp Tyr Gin His Arg Arg Gly Gin Ala Pro 40 Gly Val Ser Asn Arg Ala Gly Gly Val Pro Asp Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 70 75 Asp Phe Ala Ile Tyr Tyr Cys Gin Thr Tyr Gly Phe Gly Gin Gly Thr Lys Val Asp Ile Lys Arg 100 105 INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear :'i i~ ii :i i r i i

I:

i- 1 aL<2~ AwZAA,. ;r I vv.~ 250 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ii9: Giu Leu Thr Gin Ser Pro dly Thr Leu Ser Leu Thr Pro Gly Glu Arg 1 5 10 Ala Ile Leu Ser Cys Lys Thr Ser His Asn Ile Trp Ser Arg Arg Leu 25 Ala Trp Tyr Gin Leu Lys Ser Gly Gin Ala Pro Arg Leu Leu.Ile Tyr 40 Giy Vai Ser Lys Arg Ala Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser *50 55 Gly Ser Aia Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Giu Pro Giu 70 75 .Asp Phe Ala Vai Tyr Tyr Cys Gin Thr Tyr Cly Gly Ser Ala Tyr Thr 90 Phe Gly Gin Gly Thr Lys Leu Asp Ile Lys Arg 100 105 INFORMATION FOR SEQ ID NO:120: i) SEQUENCE CHARACTERISTICS: LENGTH: i07 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Thr Pro Gly Giu Arg 1 5 *10 Ala Ile Leu Ser Cys Lys Thr Ser His Asn Ile Trp Ser Arg Arg Leu 25 Ala Trp Tyr Gin Leu Lys Ser Gly Gin Ala Pro.Arg Leu Leu Ile Tyr 40 Gly Vai Ser Lys Arg Ala Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser 55 251 Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Arg Val Glu Pro Glu 70 75 Asp Phe Ala Val Tyr Tyr Cys Gin Thr Tyr Gly Gly Ser Ala Tyr Thr 90 Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg 100 105 INFORMATION FOR.SEQ ID NO:121: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Ser Thr Pro Gly Glu Arg 1 5 10 Ala Ile Leu Ser Cys Lys Thr Ser His Asn Ile Trp Ser Arg Arg Leu 25 Ala Trp Tyr Gin Val Lys Ser Gly Leu Pro Pro Arg Leu Leu Ile His 40 0 0.

Gly Val Ser Arg Arg Ala Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 Gly Ser Ala Arg Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Ala 70 75 Asp Phe Ala Val Tyr Tyr Cys Gin Thr Tyr Gly Gly Ser Ser Tyr Ser 90 Phe Gly Gin Gly Thr Lys Leu Asp Phe Asn Arg 100 105 INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 107 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein -252 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu 1 5 10 Ala Val Leu Ser Cys Arg Thr Ser Arg Asn Ile Ala Trp Tyr Gin Val Arg Arg Gly Gin Ala Pro 35 Gly Val Ser Lys Arg Ala Gly Gly Val Pro Asp 50 55 Gly Ser Ala Arg Asp Phe Thr Leu Thr lie Ser 65 70 Asp Phe Ala Val Tyr Phe Cys Gin Thr Tyr Gly 85 90 Phe Gly Gin Gly Asn Lys Leu Asp Ile Arg Arg 100 105 INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Asn Trp Arg Arg Arg Gly Glu Arg Ile Gly Pro Tyr (xi) Gin 1 Ala Phe Met Phe SEQUENCE DESCRIPTION: SEQ ID NO:123: Val Lys Leu Leu Glu Gin Ser Gly Ala Glu Val Lys Lys 5 Ser Val Lys Val Ser Cys Gin Ala Ser Gly Tyr Arg Phe 25 Val Leu His Trp Ala Arg Gin Ala Pro Gly His Arg Pro 40 Gly Trp Ile Asn Pro Ala Asn Gly Val Thr Glu Ile Pro 55 Gin Asp Arg Val Ser Leu Thr Arg Asp Thr Ser Ala Gly 70 75 Gly Asn Trp Lys Val 253 Tyr Leu Glu Leu Thr Asn Leu Arg Phe Ala Asp Thr Ala Val Tyr Tyr 90 Cys Ala Arg Val Gly Glu Trp Thr Trp Asp Asp Ser Pro Gin Asp Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val 115 120 125 INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: Gin Val Lys Leu Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 Ala Ser Val Lys Val Ser Cys Gin Ala Ser Gly Tyr Arg Phe Ser Asn 25 Phe Val Leu His Trp Ala Arg Gin Ala Pro Gly His Arg Pro Glu Trp 40 Met Gly Trp Ile Asn Pro Ala Asn Gly Val Thr Glu Ile Ser Pro Lys 50 55 Phe Gin Asp Arg Val Ser Leu Thr Gly Asp Thr Ser Ala Ser Thr Val 70 75 Tyr Leu Glu Leu Arg Asn Leu Arg Phe Ala Asp Thr Ala Val Tyr Tyr 90 Cys Ala Arg Val Gly Glu Trp Thr Trp Asp Asp Ser Pro Gin Asp Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Arg Gly Thr Thr Val Thr 115 120 125 INFORMATION FOR SEQ ID NO:125: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear I 1 254 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125: Gin Val Lys Leu Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 Ala Ser Val Lys Val Ser Cys Gin Ala Ser Gly Tyr Arg Phe Ser Asn 25 30 Phe Val Leu His Trp Ala Arg Gin Ala Pro Gly His Arg Pro Glu Trp 40 Met Gly Trp Ile Asn Pro Ala Asn Gly Val Thr Glu Ile Ser Pro Lys 50 55 Phe Gin Asp Arg Val Ser Leu Thr Gly Asp Thr Ser Ala Ser Thr Val 70 75 Tyr Leu Glu Leu Arg Ser Leu Arg Phe Ala Asp Thr Ala Val Tyr Tyr 90 Cys Ala Arg Val Gly Giu Trp Thr Trp Asp Asp Ser Pro Gin Asp Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val 115 120 S2) INFORMATION FOR SEQ ID NO:126: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126: Gln Val Lys Leu Leu Glu Gn Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 Ala Ser Val Lys Ile Ser Cys Gin Ala Ser Gly Tyr Arg Phe Thr Asn 25 Phe Val Leu His Trp Ala Arg Gin Ala Pro Gly Gn Arg Pro Glu Trp 40 -255 met dly Trp Phe Asn Pro Ala Asn dly 55 Phe Gin Asp Arg Val Ser Phe Thr Gly 70 Tyr Val Glu Leu Arg Asn Leu Arg Ser Cys Ala Arg Val Gly Pro Trp Thr Trp 100 105 Tyr Tyr Met Asp Val Trp Gly Lys Gly 115 120 INFORMATION FOR SEQ ID NO:127: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein.

Giu Ile Ser Pro Lys Ser Ala Ser Thr Ala Thr'Ala Val Tyr Tyr Ser Pro Gin Asp Asn 110 Val.

(xi) Gin Ala Phe Met ?he Tyr Cys Tyr SEQUENCE DESCRIPTION: SEQ ID NO:127: Val Lys Leu Leu Glu Gin Ser Gly Ala Ser Val Lys Val Ser..-Cys Gin Ala Ser 20 Val Leu His Trp Ala Arg Gin Ala Pro 40 Gly Trp Ile Asn Pro Ala Asn Gly Val Gin Asp Arg Val Ser Leu Thr Gly Asp Leu Glu Leu Arg Asn Leu Arg Phe Ala Aia Arg Vai Gly Giu Trp Thr Trp Asp 100 105 Tyr Met Asp Vai Trp Gly Lys Cly Thr 115 120 Lys Arg Arg Ile Ala Ala Pro -256 INFORMATION FOR SEQ ID NO:128: SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein xi) SEQUENCE DESCRIPTION: SEQ ID NO:128: Gin Val Lys Leu Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly

S

1 5 10 Ala Ser Val Lys Leu Ser Cys Gin Ala Ser Gly Tyr Arg Phe Ser Asn 25 Phe Val Leu His Trp Ala Arg Gin Ala Pro Gly His Arg Pro Glu Trp 35 40 45 Met Gly Trp Ile Asn Pro Ala Asn Gly Val Thr Glu lie Ser Pro Lys 55 Phe Gin Asp Arg Val Ser Leu Thr Gly Asp Thr Ser Ala Ser Thr Val 70 75 Tyr Leu Glu Leu Arg Asn Leu Arg Phe Ala Asp Thr Ala Val Tyr Tyr 85 90 95 ,i Cys Ala Arg Val Gly Glu Trp Thr Trp Asp Asp Ser Pro Gln Asp Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr 115 120 125 INFORMATION FOR SEQ ID NO:129: Ii SEQUENCE CHARACTERISTICS: LENGTH: 125 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129: Gin Val Lys Leu Leu Glu Gin Ser Gly Thr Glu Val Lys Lys Pro Gly 1 5 10 S~ -257- Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asn .25 Phe Pro Leu His Trp Val Arg Gln Ala Pro Gly Gin Arg Pro Glu Trp 40 Met Gly Trp Ile Lys Ile Val Asn Gly Giu Lys Lys Tyr Ser Gin Lys 55 Phe Val Asp Arg Val Thr Phe Thr Gly Asp Thr Ser Ala Asn Thr Ala 70 75 Tyr Met Giu Val Arg Gly Leu Arg Ser Ala Asp Thr Ala. Thr Tyr Tyr *Cys Ala Arg Val Gly Giu Trp Thr Trp Asp Met Asp Pro Gin Ala Asn 100 105 11.0 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr 115 120 125 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY:. linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Gin Val Lys Leu Leu Glu Gln Ser Gly Ala Glu Val Lys Lys Pro Gly 1 .5 i15 Ala Ser Val Lys Val Ser Gys Gin Ala Ser Gly Tyr Arg Phe Ser Asn 25 Phe Val Ile His Trp Val Arg Gln Ala Pro Gly Gin Arg Phe Glu Trp 40 Met Gly Trp Ile Asn Pro Tyr Asn Gly Asn Lys Clu Phe Ser Ala Lys 55 Phe Arg Asp Arg-Vai Thr Phe Thr Ala Asp Thr Asp Ala Asn Thr Ala .70 .75 Tyr Met Giu Leu Arg Ser Leu Arg Ser Ala Asp Thr Ala Ile Tyr Tyr 90 -258 Cys Ala Arg Val Gly Pro Tyr Thr Trp Asp Asp Ser Pro Gin Asp Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val 115 120 INFORMATION FOR SEQ ID NO:131: SEQUENCE CHARACTERISTICS: S. LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131: Gin Val Lys Leu Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly e 1 5 10 Ala Ser Val Lys Val Ser Cys Gin Ala Ser Gly Tyr Arg Phe Ser Asn 25 Phe Val Leu His Trp Ala Arg Gin Ala Pro Thr Gin Asp Leu Glu Trp 40 S,. Met Gly Trp Ile Asn Pro Ala Asn Gly Val Lys Glu Ile Ser Pro Lys 50 55 Phe Gin Asp Arg Val Ser Leu Thr Gly Asp Thr Ser Ala Ser Thr Val 70 75 Tyr Leu Glu Leu Arg Ser Leu Arg Phe Ala Asp Thr Ala Val Tyr Tyr 90 Cys Ala Arg Val Gly Glu Trp Thr Trp Asp Asp Ser Pro Gin Asp.Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val 115 120 INFORMATION FOR SEQ ID NO:132: SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein i 1 259 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132: Gin Val Lys Leu Leu Glu Gin Ser Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 Ala Ser Val Lys Val Ser Cys Gin Ala Ser Gly Tyr Arg Phe Ser Asn 25 Phe Val Leu His Trp Ala Arg Gin Ala Pro Gly His Arg Pro Glu Trp 40 Met Gly Trp lie Asn Pro Ala Asn Gly Val Thr Glu lie Pro Pro Lys 55 Phe Gin Asp Arg Val Ser Leu Thr Arg Asp Thr Ser Ala Gly Thr Val 65 70 75 Tyr Leu Glu Leu Thr Asn Leu Arg Phe Ala Asp Thr Ala Val Tyr Tyr 85 90 Cys Ala Arg Val Gly Glu Trp Thr Trp Asp Asp Ser Pro Gin Asp Asn 100 105 110 Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val 115 120 INFORMATION FOR SEQ ID NO:133: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs.

TYPE: nucleic acid l STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO i (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133: TCGAGGGTCG GTCGGTCTCT AGACGGTCGG TCGGTCA 37 INFORMATION FOR SEQ ID NO:134: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid 260 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134: CTAGTGACCG ACCGACCGTC TAGAGACCGA CCGACCC 37 INFORMATION FOR SEQ ID NO:135: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135: CGGTCGGTCG GTCCTCGAGG GTCGGTCGGT CT 32 INFORMATION FOR SEQ ID NO:136: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136: -261 CTAGAGACCG ACCGACCCTC GAGGACCGAC CGACCCAGCT INFORMATION FOR SEQ ID NO:137: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137: CAAGGAGACA GGATCCATGA AATAC INFORMATION FOR SEQ ID NO:138: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138: AGGGCGAATT GGATCCCGGG CCCCC INFORMATION FOR SEQ ID NO:139: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 262 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO S(xi) SEQUENCE DESCRIPTION: SEQ ID NO:139: CTAGTCATCA TCATCATCAT TAAGCTAGC 29 INFORMATION FOR SEQ ID NO:140: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140: CTAGGCTAGC TTAATGATGA TGATGATGA 29 INFORMATION FOR SEQ ID NO:141: SEQUENCE CHARACTERISTICS: LENGTH: 13 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: peptide FRAGMENT TYPE: internal (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 1 OTHER INFORMATION: /label- J (ix) FEATURE: NAME/KEY: Modified-site LOCATION: 13 OTHER INFORMATION: /label- ZC S263 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141: Ser Ile Ser Ile Gly Pro Gly Arg Ala Phe Tyr Thr Gly 1 5 INFORMATION FOR SEQ ID NO:142: SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142: Leu Leu Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser 1 5 10 15 Leu Thr Cys Thr Val Ser Gly Gly Ser Leu Ser Ser Phe Asp Trp Asn 20 25 e Trp Ile Arg Gin Pro Ala Gly Lys Gly Leu Glu Trp Ile Gly Arg Ile 35 40 Tyr Pro Ser Gly Asn Thr His Tyr Asn Pro Ser Leu Arg Ser Arg Val 50 55 Thr Met Ser Arg Asp Thr Ser Lys Asn Gin Phe Ser Val- Lys Leu Thr 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Leu Tyr Tyr Cys Ala Arg Glu Asn 85 90 Thr Gly Arg Thr Ile Glu Glu lie Gly Asn Phe Phe Asp lie Trp Gly 100 105 110 Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 INFORMATION FOR SEQ ID NO:143: SEQUENCE CHARACTERISTICS: LENGTH: 122 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein -264 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143: Leu Leu Lys Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Val Ile Ser Ala Phe Ser Phe Ser Gly Tyr Asn Ile Asn 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Giu Trp Val Ser Ser Ile 40 *Ser Met Ser Thr Gly Ser Leu Ser Tyr Ala Asp Ser Met Lys Gly Arg 55 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr Leu Giu Met 70 75 *Ser Ser Leu Thr Ala Gi u Asp Thr Ala Met Tyr Tyr'Cys Ala Ala Arg 90 Thr Pro Leu Val Gly Arg Ala Leu Asp Ile Trp Gly Gin Gly Thr Val *:*100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 INFORMATION FOR SEQ ID NO:i44: SEQUENCE CHARACTERISTICS: LENGTH: 132 amino acids TYPE: amino acid TOPOLOGY: linear C (ii) MOLECULE TYPE: protein, (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i44: Leu Leu Giu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Ser Tyr Gly Met Asn.

25 Trp Val Arg Gin Ala Pro Gly Lys Gly Pro Giu Trp Val Ala Tyr. Ile 40 Ser Ser Ser Arg Lys Tyr Thr Giu Tyr Ala Asp Ser Val Lys Giy Arg 55 Phe Thr Ile Ser Arg Glu Asn Ala Lys Tyr Ser Val Phe Leu Gin Leu -265 70 75 Asp Ser Leu Thr Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Gly 90 Arg Asp Phe Tyr Ser Gly Phe Gly Arg Arg Asp Asp Phe His Leu His 100 105 110 Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly 130 INFORMATION FOR SEQ ID NO:145: SEQUENCE CHARACTERISTICS: LENGTH: 126 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145: Leu Leu Glu Gin Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu 1 5 10 Arg Ile Ser Cys Val Ala Ser Gly Asp .Ile Phe Tyr Ser Tyr Ala Met 25 Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Ser 35 40 lie Ser Gly Thr Gly Gly Ser Asn Tyr Tyr Ala Asp Ser Val Lys Gly 55 60 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Leu Tyr Leu Gin 70 75 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys Ala Arg 90 Asp Arg Gly Pro Arg lie Gly Ile Arg Gly Trp Phe Asp Ser Trp Gly 100 105 110 Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 INFORMATION FOR SEQ ID NO:146: 266 SEQUENCE CHARACTERISTICS: LENGTH: 124 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146: Leu Leu Glu Ser Gly Gly Gly Leu Val Gin Pro Gly Gly Ser Leu Arg 1 5 10 Leu Ser Cys Ala Ala Ser Gly Phe Leu Tyr Ser Ser Phe Ala Met Ser 20 25 Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Ala Trp Val Ser Thr Ile 35 40 Ser Ala Ser Gly Gly Ser Thr Lys Tyr Ala Asp Ser Val Lys Gly Arg.

50 55 Phe lie lie Ser Arg Asp Asn Ser Lys Asn Thr lie Tyr Leu Gin Met 70 75 Asp Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asn 85 90 Phe Arg Ala Phe Ala Arg Asp Pro Trp Gly Asp Trp Gly Gin Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Ala Ser Thr Lys 115 120 INFORMATION FOR SEQ ID NO:147: SEQUENCE CHARACTERISTICS: LENGTH: 109 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147: Met Ala Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 Glu Arg Val lie Val Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Asn 267 Tyr Leu Ala Trp Tyr Gin Gln*Lys Pro Gly 40 Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly 55 Gly Ser Gly Ser Giy*Thr Asp Phe Thr Leu 70 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin 90 Thr Phe Gly Gin Gly Thr Lys Val Ciu Ilie 100 105 INFORMATION FOR SEQ ID NO:i48: SEQUENCE CHARACTERISTICS: LENGTH: 112 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein GIn :le Chr 15 "In Lys Leu His Gin Ile Thr 75 Val Arg Ala Pro Arg Leu Leu Pro Asp Arg Phe Ser Ile Ser Arg Leu Giu Tyr Gly Ser Ser Gly Arg Thr (xi) Met Glu Arg Ile Gly Pro Tyr SEQUENCE DESCRIPTION: SEQ ID NO:148: Ala Giu Leu Thr Gin.Ser Pro Gly Thr 10 Arg Ala Thr Phe Ser Cys Arg Ser Ser 20 25 Val Ala Trp.Tyr Gin His Lys P ro Gly 40 His Gly Val Ser Asn Arg Ala Ser Gly 55 Ser Gly Ser Gly Thr Asp Phe Thr Leu 70 Glu Asp Phe Ala Leu Tyr Tyr Cys Gin 90 Thr Phe Gly Gin Gly Thr Lys Leu Glu 100 105 S er S er Ala Ser Ile Tyr Lys Leu Ser Ile His Pro Arg Asp Arg Thr Arg Gly Ala Arg Thr 110 Pro 15 Thr Leu Phe Val S er Val C ly Ar g Val Ser Glu Ser Val it

'I

.11 I t-~jt 268 INFORMATION FOR SEQ ID NO:149: SEQUENCE CHARACTERISTICS: LENGTH: 111 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149: Met Ala Glu Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 9 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Asn Gly 25 Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu 35 40

*S

Ile Tyr Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Asp Arg Phe Ser 55 Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Ala Ile Ser Arg Leu Glu 65 70 75 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gin Tyr Ala Gly Ser His 90 Thr Phe Gly Gin Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala 100 105 110 INFORMATION FOR SEQ ID NO:150:

SEQUENCE.CHARACTERISTICS:

LENGTH: 111 amino acids TYPE: amino acid TOPOLOGY. linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION:. SEQ ID NO:150: Met Ala Glu Leu Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1. 5 10 Asp Arg Val Thr lie Thr Cys Arg Pro Ser Gin Gly Ile Gly Arg Phe 25 j iLi;r jr; 4 Z ArA~.tS*I j -269 Phe Asn Trp Tyr Gin Gin Lys Pro Gly 40 Tyr Ala Ala Asp Ile Leu Gin'Ser Giy 55 *Ser Gly Ser Gly Thr Asp Phe-Thr Leu- 65 70 Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Thr Ph-e Gly Gin Gly Thr Arg Leu Asp 100 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 112 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein Lys Vai Thr Gin 90 Ile Ala Pro Ile 75 Ser Ly s Pro S er S er Tyr Arg Asn Leu Leu Ile Arg Phe Ser Gly Ser Leu Gin Pro Ser Thr Pro Tyr Thr Vai Ala 110 (xi) SEQUENCE DESCRIPTION: SEQ ID Met Ala Giu Leu Thr Gin Ser Pro Ser Ser Leu 1 5 10 Asp Arg Val Thr Ile Thr Gys Ar g Ala Ser Gin 20. 25 Tyr Leu Aia Trp Tyr Gin Gin Lys Pro Gly Gin 35 40 Ile Phe Gly Ala Tyr Ser Arg Ala Thr Gly Ile 55 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr *.70 75 Pro Glu Asp Phe Ala Val Tyr Tyr Gys Gin Gin 90 Ile Thr Phe Gly Pro Giy Thr Lys Val Asp Ile 100 105 INFORMATION FOR SEQ ID NO:i52: Ser Gly Ala Pro Ile Tyr Lys Ala Val Pro Asp S er C ly Arg Ser Vai Ser Ser 30 Arg Leu Arg Phe Arg Leu Ser Ser Thr Val 110 Gly Ser Val Ser Glu Pro Al a -4 o".

270 SEQUENCE. CHARACTERISTICS: LENGTH: 729 base pairs TYPE: nucleic acid STRANDEDNESS:,double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 9.-715 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i52: AGCTTACC ATG GGT CTG CCC ACT CAG GTC CTG CCC TTG CTG CTG CG TG Met Gly Val Pro Thr Gin Val Leu Cly Leu Leu Leu Leu Trp 0* CTT ACA CAT CCC AGA Leu Thr Asp Ala Arg TCT GAG ATC CTT Cys Glu Ile Val 20 CTC ACGCGAG TGT GCA CCC ACC Leu Thr Gin Ser Pro Gly Thr 25 ACC TTG TGG TCT AGG TGC ACT Thr Phe Ser Cys Arg Ser Ser 40 CTG TCT CTC TCT CCA CCC GAA AGA GCC Leu Ser Leu Ser Pro Gly Glu Arg Ala

S*

S

CAC..AGC.ATT

His Ser Ile GAG GGT CCA Gin Ala Pro ACC CGC CCC GTA- CC Ser Arg Arg Val Ala TGC TAC CAC CAC Trp Tyr Gin His AAA CGT. GCC Lys Pro Cly CCC TCT GC Ala Ser Cly AGG CTG GTC ATA Arg Leu Val Ile GGT CTT TCC AAT Gly Val Ser- Asn ATG TGA Ile Ser GAC AGG TTC AGC CCC ACT CCC TCT CCC Asp Arg Phe.Ser Gly Ser Gly Ser Gly GAC TTC ACT GTC 290 Asp Phe Thr Leu ATC ACC AGA GTG Ile Thr Arg Val CCT CAA GAC TTT Pro Clu Asp Phe CTC TAC TAG TCT Leu Tyr Tyr Gys GIG TAT GCT CC Val Tyr Cly Ala TGG TAG ACT TTT Ser Tyr Thr Phe

GCC

C ly 120 GAG CCC ACC AAA GTG GAG Gin Gly Thr Lys Leu Giu 125 ACG AAA GGA Arg Lys Arg CTC CGT GCA GCA Val Pro Ala Pro TGT GC Ser Val 135 TTG ATCTTC Phe Ile Phe CCGCGCA TCT Pro Pro Ser 140 271 CAT GAG GAG Asp Glu Gln 145 TTG AAA TCT Leu Lys Ser GGG ACT CCC TCT CTT GTG TGC CTG CTG AAT 482 Gly Thr Ala 150 Ser Val Val Cys 155 Leu Leu Asn AAC TTC Asn Phe 160 TAT CCC AGA GAG Tyr Pro Arg Glu AAA GTA GAG TGG Lys Val Gin Trp GTG CAT AAC GCC Val Asp Asn Ala GAG GAG AGG AAC Gin Asp Ser Lys 190 530 578 GAA- TCG CGT AAG Gin Ser Gly Asn GAG GAG ACT GTG Gin Glu Ser Val ACA GAG Thr Glu 185 GAG AGG ACC TAG Asp Ser Thr Tyr CTG AGG AGG ACC Leu Ser Ser Thr ACG CTG AGC Thr Leu Ser 0 AAA GCA GAG Lys'Ala Asp 205 GAG GGC CTG Gin Gly Leu 220 TA ATTGTACAGA TAG GAG AAA Tyr Glu Lys AGT TCG CCC Ser Ser Pro 225 AAA GTG TAG GCC Lys Val Tyr Ala CAA GTC ACC CAT Glu Val Thr His 626 674 725 CTC AGA AAG AGG Val Thr Lys Ser AAC ACG GGA GAG Asn Arg Gly Clu

TGT

Cys 235

ATTC

INFORMATION FOR SEQ ID NO:153: SEQUENCE CHARACTERISTICS: LENGTH: 235 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i53:

I

;i Met Gly Val Pro Thr Gin Val Leu Gly Leu Leu Leu Leu Trp Leu Thr Asp Ala Arg Cys Clu Ile Vai Leu Thr Gin Ser Pro Cly Thr Leu Ser 25 Leu Ser Pro Gly Clu Arg Ala Thr Phe Ser Cys Arg Ser His Ser Ile Arg Ser Arg Arg Val Pro Arg Leu Val Ile His 70 Ala Trp Tyr Gin His Gly Vai Ser Asn Arg Pro Cly Gin Ala Ala Ser Gly Ile Asp Arg.Phe Thr Arg Val Gly. Ala Ser 115 Arg Thr Val 130 Gin Leu Lys 145 Tyr Pro Arg Ser Gly Asn :..Thr Tyr Ser 195 Lys His Lys 210 Pro Val Thr 225 272 Ser Gly Thr Phe Ala Leu 105 Gly Gin Gly 120 Val Phe Ile Ser Val Val Gin Trp Lys 170 Val Thr Glu 185 Leu Thr Leu 200.

Glu Val Thr Arg Gly Glu Phe Tyr Lys Pro 140 Leu Asp Asp Lys Gin 220 Thr Cys Leu 125 Pro Leu Asn Ser Ala 205 Gly INFORMATION FOR SEQ ID NO:154: SEQUENCE CHARACTERISTICS: LENGTH: 3282 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY: CDS LOCATION: 15..452 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154: AATTCGCCGC CACC ATG GAA TGG AGC TGG GTC TTT CTC TTC TTC CTG TCA Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser 1 5 a- 27, -'Zl -273 GTA ACT ACA GOT GTG GAG TCC CAG GTT GAG CTG GTT GAG -TCC GOG GOT Val Thr Thr Gly Val His Ser Gin Val Gin Leu Vai Gin Ser Giy Ala 20 GAG GTG AAG AAG GGT 000 GGG TGA GIG AAG GTT TGT TGT GAG GGT TGT Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Gys Gin Ala Ser 35 GGA TAG AGA TTG AGT AAO TTT GTT ATT GAT TGG GIG GG GAG GG COG GlyTyr Arg Phe Ser.Asn Phe Val Ile His Trp Val Arg Gin Ala Pro .50 55 a. GGA GAG AGO TTT GAG Gly Gin Arg Phe Glu AAA OAA TTT TGA GCG Lys Giu Phe Ser Ala AGA TOG GCG AAG AGA Thr Ser Ala Asn Thr GAG AG GT GTT TAT Asp Thr Ala Val Tyr TGO ATO GGA Trp Met Gly AAO TTG GAG Lys Phe Gin GOG TAG ATG Ala Tyr Met 100 TAT TOT 000 Tyr Gys Ala TOO ATG MAT GOT TAG MGC GOA MGC Trp Ile Asn Pro Tyr Asn.Gly Asn 70 GAG AGA OTO AGO TTT ACG GO GAG Asp Arg Val Ihr Phe Thr Ala Asp 85 GAG TTG AGO AGG OTG AGO TOT GA 0Th Leu Arg Ser Leu Arg Ser Ala 105 AGA OTO 000GG A TAT AOT TOG OAT Arg Val Gly Pro Tyr Ser Trp Asp 120 ATO GAO OTO TOG 0CC AAA GGA AGO Met Asp Val Trp Oly Lys Gly Thr 242 290 338 386 434 110

TOT

Ser

OAT

Asp 125

GOG

Pro GAG GAG MAT TAT TAT Gin Asp Asn Tyr Tyr 130 140 AG GIG ATO OTO AGO TGA GCTTOGAGGA AGGGOGATO GGTGTTCGGG 482 Thr Val Ile Val Ser Ser 145

CTGGGAOGGT

GAGTAGTTGG

GACAGOTTCG

OTGOCTOGA

AAGAGGMGGG

GOMOGAGO

CAGGAAGGCA

CAGGGAGAGO

GGTGGAAOAO

OCGGMCGGT

CGGGTT

GAGCTTGO

TGGAGAAGMA

GTGAGGCTG

GGCCCGOCT

OGTCTTOTOG

GAG OTOTGO

OAGGTOTG

AGAGTGGTGA

GAGGOAGAGO

AGTTGGTGAG

GTGGCTGOAG

GGGTGTTOAG

TTTTTCOA

GO CAGAG CGG

TGGAAGTCAO

GOAGTGTAGT

TAGATGTGGA

AGGGAGGAC

GOATCCGGO

CGGAGGGGT

GGCTCTGGGC

CGGGAG

AGGTGAATGA

AGOGAGGOAG

TATOGAGGOG

AGGCAGAGGC

CCTGGTGAAG

GAGOGOCGT

CGMGGGGAOG.

GGTGTGTGOT

CGCOAGGO

GAGTCATGGT

TAGGTOGGOC

542 602 662 722 782 842 902 962

TAACCCAGGC

CCGGGAGGAC

GCTCGGACAC

CAAATCTTGT

GCCCTCCAGC

AG CCGGGTG C

CATCAGTCTT

AGGTCACATG

ACGTGGACGG

OCACGTACCC

AGTACAAGTG

AAGCCAAAGG

CCCTCTGCCC

CCACAGGTGT

ACCTGCCTGG

CAGCCGGAGA

CTCTACAGCA

TCCGTGATGC

GGTAAATGAG

ATGCTTGGCA

CAGCGCTGCC

TGAGGCCTGA

CTGTGCAGGT

GTGGGGGATT

GAAGCCCTAG

GTTCTGTGAG

CCTGCACACA

CCTGCCCCTG

CTTCTCTCCT

GACAAAACTC

TCAAGGCGG

TGACACGTCC

CCTCTTCCCC

CGTGGTGGTG

CGTGGAGGTG

TGTGGTCAGC

CAAGGTCTCC

TGGGACC CGT

TGAGAGTGAC

ACACCCTGCC

TCAAAGGCTT

ACAACTACAA

AGCTOACCGT

-ATGAGGCTCT

TCCGACGGCC

CGTACCCCCT

CTGGGCCGCT

GTdGCATGAG

GTGCCTGGGC

TGCCAGCGTT

GAG CCCCTGG

CGCCCTGTCC

AAGGGGCAGG

AC CTAAG CCC

CCCAGATTCG

ACACATGCCC

ACAGGTGCCO

ACCTCCATCT

CCAAAACCCA

GACGTGAGCC

CATAATGCCA

GTCCTCACCG

AACAAAGCCC

GGGGTGGGAG

CGCTGTACCA

CCCATCCCGG

CTATCCCAGC

GACCACGCCT

GGACAAGAGC

GCACAACCAC

GGCAAGCCCC

GTACATACTT

GCGAGACTGT

GGAGGCAGAG

CGCCTAGGGT

GCCCTCGCTC

GGACAGACAC

TCCGACCTCC

274

TGCTGGGCTC

ACCCCAAACG

AGTAACTCCC

ACCGTGCCCA

TAGAGTAGCC

CTCCCTCAGC

AGGACACCCT

ACGAAGACCC

AGACAAACC

TCCTGCACCA

TCCCAGCCCC

GGCCACATGG

AC.CTCTGTCC

GATGACCTGA

GAGATCGCCG

CCCGTGCTCG

AGGTGGCAGG

TACACGCAGA

CGCTCCCC.GG

CCCGGGCGCC

GATGGTTCTT

CGGGTCCCAG

GG GGCT GAG C

CAGCGCACC

ACAGCCCCTG

ATGCCCACTC

AGACCTGCCA

CCAA.ACTCTC

A:ATCTTCTCT

GGTAAGCCAG

TGCATCC.AGG

ACCTGAGGCC

CATGATCTCC

TdAGGTCAAG

GCGGGAGGAG

GGACTGGCTG

CATGGAGAAA

ACAGAGGCCG

CTACAGGGCA

CCAAGAACCA

TGGAGTGGGA

ACTCCGACGG

AGGGGAACGT

AGAGCCTCTC

OCT CTCGCGG

CAGCATGGAA

TCCACGGGTC

TGTCCCCACA

CAGGGGCTGC

TGCCCTGCGC

CCTCTGTAGG

GGGGGCATGC

AGAGCCATAT

CACTCCCTCA

CTGCAGAGCC

CCCAGGCCTC

GACAGGCCCO

GGGGAGGAC

CGGACCCCTC

TTCAACTGGT

CAGTACAACA

AATGGCAAGG

ACCATCTCCA

GCTCGGCCCA

GCCCCGAGAA

GGTCAGCCTG

GAGCAATGGG

CTCCTTCTTC

CTTCTCATGC

CCTGTCTCCG

TCGCACGAGG

ATAAAGCACC

AGGCCGAGTC

CTGGCCCAGG

CCTCGGCAGG

TGCCAGG

AGACTGTCCT

CTAGTCCATG

1022 1082 1142 1202 1262 1322 1382 1442 1502 1562 1622 1682 1742 1802 1862 1922 1982 2042 2102 2162 2222 2282 2342 2402 2462 2522 275- TACCCCCACC GCACTAACCC

TCCGTAGGGA

TGCCCAGCCT

CTGTGGAGGG

CCCCCGCACT

AGGGAGCCTC

TGAACACTCC

CTCTCGCCAG

GCTGCCCCTG

CTGCCCCACT

TTTGTAGAGC

AAAATGA.TC

ACCAATAGCA

TTGTCCAAAC

CAGGCCCCTCC

CGCACCCGCA

ACTCGTGCAG

CAGGTTGCC

ACCCGGCGCA

TCCGACACAG

CTTCTCCACA

CAGCCCCGAG

TCCCAGTGC

TTTTACTTC

CAATTCTTCT

TCACAAATTT

TCATCAATGT

CTCACCCATC

TGCCACACA

ATGCCCACAC

GGCCACACGG

ACTGCACAGC

GCCCCCACGA

TGCTGACCTG

ACACACACAG

GCCGTTCCCT

TTTAAAAAAC

TGTTAACTTG

CACAAATAAA

ATCTTATCAT

ACCCACTCCC

ACACACTCAG

CCACCACACA

ACCCAGACCA

GCCCCACC

CTCACACAAA

CCGATCACAC

GCAGGCCGGA

CTCCCACACC

TTTATTCCAG

GCATTTTTTT

GTCTAGATCC

GGGACATCCA

TCCAGACCCG

CACACGTGCA

GAGCAAGCTC

GCACCTCAAC

CC GAG CCCTC

ACCACGTCAC

TCATAATCAG

TCCCCCTGAA

CTTATAATGC

CACTGCATTC

CTCGCTCTCC

CTCTCGCGCC

TTCAACAAAA

CGCCTCACAC

CTCGCACAG

GCCCACGAC

CTCTCACAAG

GTCCCTGGCC

OCATACCACA

CCTCAAACAT

TTACAAATAA

TACTTGTGGT

2582 2642 2702 2762 2822 2882 2942 3002 3062 3122 3182 3242 3282 INFORMATION FOR SEQ ID NO:155: SEQUENCE CHARACTERISTICS: LENGTH:-146 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Glu Trp.Ser Trp .Val Phe Leu Phe Phe 1 5 10 Val His Ser Cmn Val Gin Leu Val Gin Ser NlO: 155: Leu Ser Val Thr Thr Gly Pro Cly Ala Val Lys Val Ser Cys 40 Ile His Trp Val Arg 55 Trp Ile Asn Pro Tyr 70 C in Gin Giy Ala Clu Val Lys Lys Ala Ser Cly Tyr Arg Phe Ala Pro Cly Gin Arg Phe Ser Asn Glu Trp Phe Val Met Cly Asn Cly Asn Lys Ciu Phe 276 Val Thr Phe Thr 90 Ala Lys Phe Gin Asp Arg Thr Ala Tyr Met. Glu Leu 100 Tyr.Tyr Cys Ala Arg Val 115 Asp Asn Tyr Tyr Met Asp 130 Ala Asp Thr Ser Ala Asn Arg Ser Leu Arg Ser Ala Asp 105 Gly Pro Tyr Ser Trp, Asp Asp 120 125 Val1 Trp Gly Lys Gly Thr Thr 135 140 Thr Ala Val 110 Ser Pro Gln Val Ile Val 0* Ser Ser 145 INFORMATION FOR SEQ ID NO:156: SEQUENCE CHARACTERISTICS: LENGTH: 13254 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156: TTCATTGATC ATTAATCAGC CATACCACAT TTGTAGAGGT TTTACTTGCT TTAAAAAACC

TCCCACACCT

TTATTG GAG C

GATTTTTTTC

TCTGGATCTC

GTCCGAAATA

TGTTTATCGC

TGAAAATGTC

TTTTGGG CAT

ACGACTTTGG

ACAGACGATA

CATATCGATC

CCCCCTGAAC

TTATAATGGT

AGTGGATTCT

TAGCTTCGTG

CGCGTTTTGA

CGATAGAGAT

GCCGATGTGA

ACGCGATATC

TGACTTGGGC

TGAGGCTATA

TATACATTGA

CTGAAACATA

TACAAATAAA

AGTTGTGGTT

TCAAGGACGG

GATTTCTGTC

GGCGATATTG

GTTTCTGTGT

TGGCGATAGC

GATTCTGTGT

TCGCCGATAG

ATCAATATTG

AAATGAATGG

GGAATAGCAT

TGTCCAAACT

TGACTGCAGT

GCCGACTAAA

GAAAAATCGA

AACTGATATG

GCTTATATCG

GTCGCAAATA

AGGCGACATC

GCCATTAGCC

AATTGTTGTT

CACAAATTTC

CATCAATGTA

GAATAATAAA

TTCATGTCGC

TATTTGAAAA

GCCATTTTTC

TTTACGGGGG

TCGCAGTTTC

AAGCTGGCAC

ATATTATTCA

GTTAACTTGT

ACAAATAAAG

TCTTATGATG

ATGTGTGTTT

GCGATAGTGG

TATGGCATAT

CAAAAGT CAT

ATGGCGATAG

GATATAGGTG

ATGGCCAATG

TTGGTTATAT

AGCATAAATC AATATTGCCT

ATTGCCCATT

CATTTATATT GGCTCATGTC

CAACATTACC

TTAATAGTAA

ATAACTTACG

AATAATGACG

GGAGTATTTA

GCCC CCCTATT

CTTATGAC

CATGCGGTTT

AACTCTCCAC

TCCAAAATGT

GGAGGTCTAT

ACGCTGTTTT

GTGCATTGGA

TAGCC CAC C CC CCCGCTTC

CCATTATTGA

TCTTTGCCAC

.CGCACTCTGI

CACCACCCTC

CTCGGGTACC

GCCCTGCTC(

GGAGGCCAG2

GGCGGTAGG(

TGGAAGACT.

AGAGTCAGA(

TCAATTACG

GTAAATGGCC

TATGTTCCCA

CGGTAAACTG

GAGCTCAATG

TTTCCTACTT

TGGCCTACA

CC CATTGAC C

CGTAACAACT

ATAAGCAGAG

GACCTCCATA

ACGCGGATTC

CCCTTGGCTI

CTCATGTTAI

CCACTCCCCI

AACTCTCTTI

ATTTTTACA(

CGCAGTGCC(

TGTTCCGGA(

CATCCCTCCj

SCTTAGGCAC.

3 TATGTGTCT r AACGCAGCG 3 GTAACTCCC GGTCArTAGT

CGCCTGCCTG

TAGTAACGCC

CCCACTTGGC

ACGGTAAATG

GGCACTACAT

TCAATGGGCG

TCAATGGGAG

CGGCCCCATT

CTCGTTTAG'I

*GAACACACCC

CCCGTGCCCA.P

CTTATGCATC

AGGTGATGG')

ATTGGTGAC(

SATTGGCTATj

GATGGGGTC'

SGCAGTTTTT,

SATGGGCTCT'

SGGGACTCAT

h GCACGATC G AAAATGAGC G CAGAAGAAC G TTGCCGTGC 277

GCATACGTTG

GCCATGTTGA

TCATAGCCCA

ACCGCCCAAC

AATAGGGACT

AGTACATCAA

GCCCGCCTCC

CTACGTATTA

TGGATAGCCC

TTTGTTTTCG

GACGCAAATG

GAACCGTCAG

GGACCGATCC

GACTGACGTA

CTATACTGTT

SATAGCTTAC

SATACTTTCCA

k TG CCAATACP r CATTTATTAI k TTAAAGATAJ I CTCCGGTAG( G GTCGCTCGG( C CACCACCAC( T CCGGGAGCGI A TGCAGOGAG .T GTTAACGGT TATCCATATC ATAATATGTA CATTGATTAT TGACTAGTTA

TATATGGAGT

GACCCCCGCC

TTCCATTGAC

GTGTATCATA

CATTATGCCC

GTCATCCCTA

TTTGACTCAC

GACCAAAATC

GGGGGTACGC

ATCGCCTGGA

AG CCTCCCG

AGTACCGCCT

TTTGGCTTGG

CTATAGCTGT

TTACTAATCC

CTGTCCTTCA

TTACAAATTC

CGTGGGATC]

SGGCGGAGCTI

SAGCTCCTTG(

SACTGTGCCG(

SGCTTGCACC(

C TCAGTTGTTI G GAGGGCACT,

TOCCGCGTTAC

CATTCACGTC

GTCAATGGGT

TGGCAAGTAC

AGTACATGAC

TTACCATGGT

CCGGATTTCC

AACCGGACTT

GTGTAGGTG

GACGCrATCC

GCCGGGAACG

ATAGACTCTA

GCTCTATACA

GGGTTATTGA

ATAACATGC

GAGACTGACA

ACATATACAA

CCACGCGAAT

CTACATCCGA

TCCTAACAGT

SACAAGCCCGT

SCTGACGCATT

3 TCTTCTGATA 3 TAGTCTGAGC 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 -1 9

AGTACTCGTT

CCTTTCCATG

ATCGACTCTA

CTGGCTCTTT

TCAGTCCGG

TGCATACAGA

TGACTOCATG

GCACAGAGTC

CCTCAGGTCT

TGATTCTCCC

GAGCTCAGCT

CTCTGCGGGC

GGTGTCCTGG

GTCCTCAGCA

CCACACCTAC

TGGTGAGAGG

CCTGCACCCA

TCTTCACCCG

TICCCCAGGC

GGGCAGGTGC

TAAGCCCACC

AGATTCGAGT

CATGCCCACC

GTCCCCTAG

TCCATCTCTC

C CTGC CGCCC

GCTCTTTTCT

GAGGATCGAT

CTCTTCTTCC

GCTGACGTGA

TTCAGTAACT

GGATGCATCA

ACCTTTACCG

GCAGACACGC

CAGGACAATT

TCCACCAACG

ACACCCCC

AACTCACCCG

CTC TACT CC C

ATCTGCAACG

CCAGCACAG

TCCCCCCTAT

CAGGCCTCTC

TCTGGCCACC

TGGGCTCAGA

CCAAACCCCA

AACTCCCAAT

CTGCCCACGT

ACTAGCCTGC

CCTCACCACC

GCGCCACCAG

CCAGTCACCG

CCCCGGCGA

TCTCACTAAC

AGAAGCCTGG

TTGTTATTCA

ATCCTTACAA

CCCACACATC

CTCTTTATTA

ATTATATCCA

CCCCATCCCT

TGGCCTGCCT

CCCTCACCAG

TCACCACCT

TCAATCACAA

CAGGCACCCT

GCAGCCCCAG

CCCGCCCCAC

CACACOCTAC

CCTGCCAAGA

*AACTCTCCAC

CTTCTCTCTG

*AAGCCAGCCC

ATCCACCCAC

TCAGCCC

278

ACATAATAC

rCCTTGACAC

GCTCGAATTC

TACAGGTCTC

GGCCTCACTG

TTGCCTGCC

CGGAAACAAA

CGCGAACACA

TTGTCGACA

CCTCTGCCCC

CTTCCCCCTG

CCTCAAGCAC

-CGCCTGCAC

CCTCACCGTG

CCCCAGCAAC

GTCTGCTCCA

TCCAGGGCAG

TCATGCTCAG

CTC CCC CTAA

CCCATATCG

TCCCTCACCT

CACAGCCCAA

AGCCTCGCC

AGCCCCAGC

CCAGGACCAT

TGACACACTA

CAACCTTCG

CCCCCACCA

GACTCCCAG

AAGGTTTCTT

CACGCCCCGC

GAATTTTCAC

GCCTACATCC

CTCCGCCAT

AAACGAACCA

GCACCCTCCT

TACTTCCCCC

ACCTTCCCCC

CCCTCCACCA

ACCAAGCTCC

ACCCAGGCTC

CAAGCCACGC

CCAGACCCTC

CCCACCCCCT

GCAGCACCCT

CCGACACCTT

ATCTTCTCAC

CTCCACCTCA

CCCGTGCTCA

CACTCTTCCT

ACAGACTCTT

CTGCAGCTCC

TGCAATGCAG

TTCACCTCCT

CTCACGCTTC

GACAGAGCTT

CGAAGTTCCA

ACTTCACCAC

ATACTTCCCA

CCCTCATCCT

CCAACACCAC

AACCGCTGAC

CTGTCCTACA

CCTTCCGCAC

ACAAGAAAGT

AGCCCTCCTC

CCCGTCTCC

TTCTCCCTTT

CCACACAAAC

CCCCCTGACC

CTCTCCTCCC

AAAACTCACA

AGO CGGACA

CACCTCCACC

CTTCCCCCCA

2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 AAACCCAAGC ACACCCTCAT CATCTCCCCC ACCCCTCACG ICACATGCGT COTOCTOCAC 4 S~ 279 AAGACCCTGA GGTCAACTTC

AACTGGTACC

TGGACGGCGT GGAGGTCCAT 3900

CTGACCCAGG

0 0 0* 0 0**0 0

AATGCCAACA

CTCACCGTCC

AAAGCCCTCC

G EGCGAGGC

TGTACCAACC

ATCC COGGCGAT

TCCCAGCGAC

CACCCCTCCC

CAAGAGCAGG

CAACCACTAC

AAG CC CCCG C

CATACTTCCC

ACACTGTAI

GGCACAGCGC

CTAGCGTGG(

CTCCCTCCA(

CAGACACA~i GAC CTC CAT

ACCCATCTA

GGACACAAC

CCCACACAC

CACACGCC

GCACAGCAC

CAAAGCCGCG

TGCACCAGGA

CAGCCCCCAT

CACATCCACA

TCTGTCCCTA

CACCTGACCA

ATCGCCCCTG

GTCCTCGACT

TGCAC CACO

ACGCACAACA

TCCCCGCCT

GGCGCCCAC

CCTTCTTTCC

GTCCCACTCI

CCTCAGCCAC

CAGCACCTCC

k GCCCCTGCC' C; CCCACTCGC( C CCCCACGCC C GACTCCG'G A CACTCACTC A CCACACACA C CACACCACA

CTCGCTGAAT

CGAGAAAACC

GAGCCGCT

CACGGCAGCC

AGAACCAGGT

ACTGGGAGAG

CCCACGGCTC

CGAACCTCTT

GCCTCTCCCT

GTCGCGG TCG

CATCCAAATA

GGCAAGGAGT

ATCTCCAAAG

CCGCCCACCC

CCGAGAACCA

CAGCCTGACC

CAATGGGCAG

CTTCTTCCTC

CTCATGCTCC

GTCTCCGCCT

CACCAGGATG

*AAGCACCCAC.

ACAAGTGCAA

CCAAAGGTGG

TCTGCCCTGA

CGGTGTACA

TGCCTGGTCA

CCGGAGAACA

TACAGCAAGC

GTCATGCATG

AAATGACTGC

GGTCTCCAAC

GACCCGTGGG

GAGTGACGGC

CCCTGCCCCC

AAGGCTTCTA

ACTACAAGAC

TCACCGTGGA

AGGCTCTGCA

GACCCGC

GGACCACCAG TACAACAGCA CGTACCOTOT

GGTCAGGGTG

CTTGGCACCT ACCCCCTGTA ACGGGTCAGG CCGAGTCTCA CCC CACACTG CCC CTG CCCT

CCTGCCTG

CTGTACGAGA

CCCATCCCTA

CTAACCCCTG

ACATCCACTC

ACACCCGTTC

CCCCACCCTC

CGGCACGGTG

GCCACGGCAA

CTCTCCTGTT

GTCCATGTGC

CCTGTCCTGG

TCCGGCCCTG

AACAAAACCC

CGCTGCCCTG

GGCCTGACTG

TGCAGCTGTG

CGGGATTTC

GCCCTAGCAG

CTGTGAGCGC

GTAGGCACAG

CCACCTCC

TGGAGGGACT

CCGCACTGAG

GGCCCCTGCG

GCATCAGGGA

C CTGGG CCCC

CAGCGTTGCC

CCCCTGGGGA

CCTGTCGTCC

GCCCTCCCTC

ACCCGCATGG

GGTCCAGATG

GTTGGCCGG.C

CGGGCGAACT

3960 4020 4080 4140 4200 4260 4320 4380- 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220

F;

ACGTGCACGC CTCACACACG GAGCCTCACC CAACGTCCTC CCACACGTGA ACACTCCTCC GACACAGGCC

CCCACGAGCC

TGACCTCCTC

CCACGCGCCA

AGACAAACCC

CCTCAAGGCC

AGCCCTCCTC

CACGAGCCTC

TCACAAGCT

TCGGCAGCTT

GCCCCTGCAG

CTCCAGATGC

CCGCCACACA

5280 5340 5400 a a.

a. S aaa 9 CACACAGOCO ATCACACACG ACGTCACGTC CTTCCCTGCA GGGCGGATCA TAATCGCCA AAAAAACCTC CCACACCTCC CCCTGAACCT TAAGTTGTTT ATTGCAGCTT ATAATGGTTA AAATAAAGCA TTTTTTTCAC TGCATTCTAG TTATCATGTC TAGATCCTCT AGGCCGGACG TGCGGTTGGT GGCGCCTATA TCGCCGACAT CGGGGTCATG AGCCTTGTT TCGCGTGGG TTGGGCGCCA TCTGGTTCA TGCACGATTC CTACTACTGG GCTGCTTCCT AATGCGGAG GCGTTGCTOG CGTTTTTCCA TAGGCTCCGC TCAAGTCAGA GGTGGCGAAA CCCCACAGGA AGCTCCCTCG TGCGCTCTCC TGTTCCCACC CTCCCTTCGC GAAGCGTGGC GCTTTCTCAA TAGCTCGTTC GCTCCAAGCT. GGGCTGTGTO GCCTTATCCG CTAACTATCC TCTTGAGTCC GCAGCAGCCA CTGGTAACAG.GATTAGGAGA TTGAAGTGGT GGCGTAACTA GGGCTACACT CTGAAGCGAG TTACCTTCGG AAAAAGAGTT GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAAGAAGATC CTTTGATCTT TTCTACGGGG TAAGGGATTT TCGTCATGAG ATTATCAAAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TGCTTAATCA GTGAGCCACC TATGTCAGG TGACTCCGGG TCGTGTAGAT AACTACGATA GCAATGATAC CGCGAGACCC ACGCTCACCG 280

CGTGGCCCTG

TACCACATTT

GAAACATAAA

CAAATAAAGC

TTGTGGTTTG

CATCGTGCC

CACGGATGGG

TATGGTGGCA

CTTGGGGCGG

TCGCATAAGG

CCCCCTGAG

GTATAAAGAT

CTGCCGCTTA

TGCTCACGCT

,GACGAACCCC

AACCCGGTAA

GCGAGGTATG

AGAAGGACAG

GGTAGCTCTT

CAGGAGATTA

TCTGACGCTC

AGGATCTTGA

TATGAGTAAA

ATCTGTCTAT

CGGGAGGGGT

GCTCCAGATT

GCCCACTTCC

GTAGAGGTTT

ATGAATGCAA

AATAG CAT CA

TCCAAACTCA

GGCATCACCG

GAAGATCGGG

GGCCCGTGC

CGGTCCTCAA

GAGAGCGT CG

AGCATCACAA

ACCAGGCGTT

CCGGATACCT

GTAGGTATCT

CCGTTCAGCC

GACACCACTT

TAGGOCGTOG

TATTTGGTAT

GATCCGGCAA

CGCGCAGAAA

AGTGGAACGA

CCTAGATCCT

CTTGGTCTGA

TTCGTTCATC

TACCATCTGG

TATCAGCAAT

CAGTGCCGCC

TACTTGCTTT

TTGTTGTTGT

CAAATTTCAC

TCAATGTATG

GCGCCACAGG

CTCGCCACTT

CGGGACTG

CGGCCTCAAC

AC CTCGGG CC

AAATCGACGC

TCCCCCTGGA

GTCCGCCTTT

CAGTTCGGTG

CGACCGCTGC

ATCGGCACTG

TACAGAGTTC

CTGCGCTCTG

ACAAACCACC

AAAAGGATCT

AAACTCACGT

TTTAAATTAA

CAGTTACCAA

CATAGTTGCC

CCCCAGTGCT

AAACCAGCCA

5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 to GCCGGAAGGG CCGAGGCAG AAGTGGTCCT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT

S

S

S.

S S S. S

S

S S

S

SS

S

S

GCCATTGCTA

GGTTCCCAAC

TCdTTCGOTC

ATGGCAOCAC

CGTGAGTACT

CCGOCGTCAA

GGAAAACGTT

ATGTAACCCA

GGTOAGCAA

TGTTGAATAC

CTCATGAGCG

ACATTTCCCC

TATAAAAATA

GTTCCGTGGC

GTGOOCCTTT

GCGGCTTTGG

GTCCCCAGGC

CAGGCTCCCC

TcCCCGCcI

CCCATGGCTC

'CATTCCAGAt CG CCAC OG(

AACATCAAG(

CAGGCATCGT

GATCAAGGCO

CTC COAT CGT

TGCATAATTC

CAACCAAGTC

CACGGGATAA

CTTCGGGGCG

CTCGTGCACC

AAACAGOAAG

TCATACTCTT

GATACATATT

GAAAAGTGCC

GOCGTATCAC

ACCOAGOACA

CTGCGTTTAT

CAGCCAAOCT

TCCCOAGCAG

AGGAGGCAGA

AACTCCGCCC

ACTAATTTTI

GTAGTGAGGA

TCAOACACC

AAATGTACT']

GOTGTCACGC

AGTTACATGA

TGTCAGAAGT

TCTTACTGTC

ATTCTGAGAA

TACG CCCCA AAAACT CT CA

CAACTOATCT

GCAAAATGCC

CCTTTTTCAA

TGAATGTATT

ACCTGACOTC

GAGGCCCTGP

ACCCTCAAOP

AAGOAGACAC

AGATCCGGCI

GCAOAAGTA'.

AGTATGCAAj

ATCCCGCCC(

TTTATTTATI

GOCTTTTTT,

TTCCACCAT'

GTGCCTGCC

281

GCAACTTTAT

TCGCCAGTTA

TCGTGTTTG

TCCCCCATGT

AAOTTGOCCG

ATGCOATCCG

TACTGTATGC

CATAGCAGAA

AGGATCTTAC

TCAGCATCTT

OCAAAAAAGG

*TATTATTGAA

TAOAAAAATA

TAAGAAACCA

TGGCTCTTTG

GAAAATGTAA

TTTATOTTTA

GTOOGAATGTG

C' GCAAAGCATG k. OCATOCATCI

STAACTCCGCC

3 CAGAGOCCGP 3 OAGGCCTAGC 0 GCCACCTCA( CCGCCTCCAT C ATATTGCG C GTATOOCTTC A TGTGCAAAAA P CAGTGTTATC I TAAGATOCTT I GOCGACCGAO

CTTTAAAAOT

CGCTGTTGAG

TTACTTTCAC

GAATAAGGC

OCATTTATCA

AACAAATAGG

TTATTATCAT

CGOGAGCOAT

TCACACTOOC

AGAAGCTTGG

TGTCAGTTAG

CATCTCAATT

CAATTAGTCA

CAOTTCCGCC

GGCCGCCTCG

*CTTTTGCAAA

CAAGTTCCCA

CAGTCTATT

AACGTTGTT

~TTCAGCTCC

LGCOOTTAGC

~CTCATGGTt

'TCTOTGACT

~TOCTCTTGC

;CTCATCATT

k.TCCAGTTCG

GAGCOTTTCT

GACCGAAA

GGGTTATTGT

GGTTCCGCGC

GACATTAACC

CGTTCOTAAT

TCACCTTCGG

TAAATTCCTT

GGTGTGGAAA

AGTCAGCAAC

GCAACCATAG

CATTCTCC

GCCTCTOAGC

AACTAGCTTG

CTTOAACAAA

CATGTATATC

7020 7080 7140 7200 7260 73 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 CAGGGTGAGA AAGTCCAAGC TGTTGATO OTACTGGAGA AGGACTGCOC TGCAAAACGC GOACCOTOGA CTGTGAGCCC 8520 AAGTCTCTAG AACAGTTACC TGACTGGAAT

S.

6 e ee.e 0@ 6 S

S

06

S

5* e

CS

i.e.

eec.

S. S 5.5.

e e 0 .5.5 S S Oe 6 See S e ecee C ee C e e eeee.e 5 .e

GGCTCCAACA

GATCCCAACA

AATTTAACC

GCA AT GGAAC

AATGCCTTTC

CGCAGGGATA

GCAACAAATG

ATCCGCATGC

TTGCGGTAAT

CCCATACCAA

ACGCCATCCA

GCCGCCTGCA

TTTCTGCTGC

ACAACAAAGG

CAGAAGC CAT

AAAACTAATT

ACAGATCTTT

AGAGATTTAA

GATTCTAATT

GTGG AATGCC

TGAGCCTACT

CCCCAAGCAC

AACTCTTCCT

AATTATGGAA

ACTGTTTTTT

GTGACATCTA

AGCTCCTCTT

ACTCGTCTAA

AGGACTATAC

CTGCGCCCCA

TCGTGGACGC

CTGAGCTCAT

GAGATCATCT

AGCAACCTTT

CTTTAGCACd

GAAACTAAC

CAATGCCCGT

TGTCGCCAAT

TTACTTTGAA

CGTCCGCACA

AGACTTTCAG

CTGAAGGAAC

ACCTCTAAGG

GTTTGTGTAT

TTTAATGAGG

GCTGACTCTC

TTTCCTTCAG

TGCTTTGCTA

AAATATTCTG

CTTACTCCAC

T CT CAG C CCT

CTCTGAAGTT

ACCCATAATC

TCTCATCCCA

ACCTCCCTAT

TCACTACCC

CCCTCCCCAC

CTCCCTGCC

CACCCCAAC

AAGCCCATC

AACCGCCACC

GCTCTCACTC

CGCACTCCCA

CACCCGCCC

TGCCTTCTCA

TCATCTTCAC

CTTACTTCTC

TAAATATAAA

TTTACATTCC

AAAACCTCTT

AACATTCTAC

AATTCCTAAG

TTTACACCAC

TAACCTTTAT

ACACOCATAC

-282

TTTCATCCCT

CTTCCCATGT

TTCAACTACA

CACATCCTGA

ACACATCCCC

TACTCTCGTC

CCCTCCTTCT

TCCCAACTCC

CCTTTCATCT

CCATTCCTCC

CCCACCACAA

CCTACCACAT

CCTTCCACCA

CCATCCGCAT

CCTCTCCCAA

ATCACACTCC

CCTTTCCTAC.

TCCTCTCACA

ATTTTTAACT

AACCTATCCA

TTCCTCACAA.

TCCTCCAAAA

TTTTTTCACT

AAACCAAAAA

AACTACCCAT

ACTCTCTGCT

TTCGCCACCC

ACCGCAACC

GCAACCACCA

ACCCTTTTCC

TCGCCACA

ATCCTGCCCT

AAATAGCACC

TCATCCAGTA

GAACTCCAAT

TCCTCTCAAC

TCCAGCCTAC

AACCTCCAAC

TCCCCCCACT

TTCTGACCCC

CCACCACCC

TTCATCCCAC

TAATTCCACA

CTATAATCTC

ACTCATCAAT

CAAATCCCAT

AAGAACAGAA

CATCCTCTCT

GCTCCACTC

AACACTTATA

ATTAATAACT

CTTCCGCACA

TCCACAGACC

CCCCTCCTTT

TTGCCCTTCC

CAAAdCCTAT

CAACATTACA

CTCTCAACCA

TGTCAAGACT

CCTCCAGCCT

CACATCGACC

CATCCCAACC

AT CAACCA CT

GTCCCCCACC

TTTCCACTCA

TTCCAATACA

CCCCCCCAG

AACTACCTAC

TTAAACTACT

CCCAGCACTG

CTACTCATCA

ACCTACAACA

TTACTAATAC

TATACAACAA

ATCATAACAT

ATCCTCAAAA

CTACTACCTT TCACTCTCAC 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780.

9840 9900 9960 10020 10080

'I.

~4j

ATTGTCTAC

TGCCTTCACT

AAAAC CT CC C

ACTTGTTTAT

ATA AAGCATT

ATCATGTCTG

CTCTTTGTCC

TACTGGTGTT

GCATATTGAA

AGTCATTTTT

CCATAGACCA

TACOTOACAC

CCAATCCATA

TTATATACCA

TATCTACATT

TACTTATTAA

CCTTACATAA

GACCTCAATA

ATOOCTOGAC

-AACTACGCCC

CATGACCTTA

CATCCTCATC

ATTTCCAACT

CCACTTTCCA

ACOCTOCCAG

TTTACCTTTT

ACACATCATA

ACACCTCCCC

TCCACCTTAT

TTTTTCACTC

CAT CTCTACC CAAATA CGCG T ATCGCCCAT

AATCTCCCC

CCGCATACC

CTTTCCTCAC

ACCATATCAC

TCCATCTATA

TAAATCAATA

TATATTOOCT

TACTAATCAA

CTTACCCTAA

ATCACCTATC

TATTTACCCT

CCTATTCACC

TCCCACTTTC

CCCTTTTGC

CTCCACCCCA

AAATCTCCTA

CTCTATATAA

TAATTTCTAA

ATCACCCATA

CTCAACCTCA

AATCCTTACA

CATTCTACTT

TTCCTCTCAA

TTTTCACATT

ACACATGCC

ATCTCACTTT

CATATCTGC

TTCCCCGATT

CCTATATCCC

CATTCAATCA

TTCCCTATTC

CATCTCCAAC

TTACCCCCTC

ATCGCCCCC

TTCCCATACT

AAACTCCCCA

TCAATGACGC

CTACTTCCCA

ACTA CAT CAA

TTCACCTCAA

ACAACTCCC

CCACACCTCC

283 kGGCCCTTAAT

CCACATTTGT

kACATAAAAT

AATAAAGCAA

GTCCTTTCTC4

GCGACCGTCAC

TCTCTCCCC

ATATTGCAAA

CTCTCTAACT

CATACCCTT

CTCTCTCTCC

CCATAGAGGC

ATATTGCCCA

CCCATTCCAT

ATTACCCCCA

ATTACTTCAT

TCCCTCACCC

AACC CCAATA

CTTGCCAGTA

TAAATGCCCC

CTACATCTAC

TGCCCCTCCA

TGCGACTTTG

CCCATTCACG

TTTAGTCAAC

kACCAATATT

%CAGGTTTTA

C,6ATGCAATT

TACCATCACA

CAAACTCATC

TCCACTCAAT

ACTAAATTCA

AATCCATATT

CATATCCCCA

ATATCCTTTA

CAAATATCCC

CACATCAACC

TTACCCATAT

ACCTTCTATC

TCTTCACATT

ACCCCATATA

CCCAACCACC

CC CACTTTC C

CATCAACTCT

CCCTCGCATT

CTATTACTCA

TAG CGGTTTC

TTTTCCCACC

CAAATCCCCC

CCTCAGATCC

rCATCTATAC C TTCCTTTAA

GTTCTTCTTA

AATTTCACAA

AATGTATCTT

AATAAAATCT

TCTCGCCCA

TCAAAATATC

TTTTTCCAAA

CGCCCCATCC

ACTTTCCATA

TGCCACATCC

TATTCATTCC

CATATCATAA

CATTATTCAC

TCCACTTCCC

CCCCGCC CATT

ATTCACCTCA

ATCATATCC

ATCCCCACTA

TCCCTATTAC

ACTCACCCCC

AAAATCAACG

CTAGCCTCT

CCTCCACACC

10140 10200 10260 10320 10380 10440 10500 10560 10620 10680 10740 10800 10860 10920 10980 11040 11100 11160 11220 11280 11340 11400 11460 11520 11580 44 CCATCCACCC TCTTTTCACC TCCATACAAC ACACCOCCAC CCATCCACCC TCCCCCC 11640 GGAACGOTGC ATTGGAACGC AGTCTATAOO CCCACCCCCT TATACACCCC COCTTCCTCA TATTOACCAT TATTOACCAC CaGGCTCTT TGCCACAACT CTOACACGOA CTCTOTATTT ATACAACACC ACCGTCCCCA OCGAATCTCG GCTACOTOTT ATCCOAGCCC TGCTCCCATG AACAOTOOAO OCCAGACTTA OOCCGTGOCG GTAGGOTATG CGCATTTOGA AGACTTAAG CTOATAAGAG TCAOAOOTAA

CTGAGCAGTA'CTCGTTGCTO

ACTGTTCCTT TCCATGGOTC GTOTGCCCAC TCAOOTCCTO TCGTTCTCAC GCAOTCTCCA CCTOTAGOTO CAGTCACAGC GCCAOGCTCC .AAOGCTOOTC GGTTCAGCGG CAGTGGCTCT AAGACTTTOC ACTGTACTAC OOACCAAACT OGAGAOOAAA CTGATGAOCA CTTGAAATCT COAGAGACOC CAAAGTACAG AGACTGTOAC AGAGCAGGAC TGAOCAAAGC AGACTACGAG

GGATTCCCCG

TGGCTTCTTA

TGTTATAGGT

TCCCCTATTG

CTCTTTATTG

TTACAGGATG

GTGCCCGCAO

CCOOACATGO

CCTCCAGCGA

COCACACCAC

TOT CTGAAAA

CGOCGOCACA

CTCCCGTTGC

CCGCGCGC

TTTTGTG GAG

GGGTTGCTGC

GGCACCCTGT

ATTGGCAGCC

ATACATGGTG

GGGACAGACT

TGTCAGGTCT

CGAACTOTGC

GGGACTG OCT

TOOAAGOTGG

AOOAAOOACA

AAACACAAAG

284

TOCCAAOAGT

TGCATOCTAT

GATOOTATAG

GTGACCATAC

GCTATATGCC

OOGTCTCATT

TTTTTATTAA

GCTCTTCTOG

CTCATOOTCG

GATOOCCACO

TOAGOTOCG

AGAAOATGCA

GOTOCTOTTA

CACCAGACAT

TOACCOTOCT

TGCTOTGGCT

CTCTGTCTCC

O C COC TAG C

TTTOCAATAG

TCACTCTCAC

ATOOTOOCTO

CTOCACCATG

CTOTTOTGTG

ATAACGC CCT

OCACCTACAG

TCTACGCCTG

GACGTAAGTA

ACTGTTTTTG

CTTAGCCTAT

TTTCCATTAC

AATACACTCT

TATTATTTAC

ACATAACGTO

OCTACC

CTCOOCAOCT

ACCACCACTO

GAG CGOOCTT

GOCAGOTGAG

ACOOTOGAGO

AATAGCTGAC

TGACACGAAG

TACAGATOCO

AGGGGAAAGA

CTOGTACCAO

GCOTOTOC

CATOACCAGA

OTOOTACACT

TOTCTTCATC

CCTGCTGAAT

CCAATCGGGT

CCTCAGCAOC

CGAAGTCACC

CCGCCTATAO

OCTTGOOOTC

AGOTOTGOT

TAATCCATAA

CCTTCAOAOA

AAATTCACAT

CCATCTCCAC

GAGOTTOTAC

COTTOCTOCT

TGCCGCACAA

OCACCOCTOA

TTGTTCTOTT

GCAOTOTAOT

AGACTAACAO

OTTACCATOG

ACATOTOAGA

GCCACOTTOT

CACAAACCTG

ATOTOAGACA

GTOGAGCCTG

TTTOOCCAOO

TTCC COC CAT

AACTTCTATC

AACTCCCAGG

ACCOTOACOC

CATCAGOOCC

11700 11760 11820 11880 11940 12000 12060 12120 12180 12240 12300 12360 12420 12480 12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 285 TGAGATCGCC. CGTCACAAAG ACCTTCAACA GGGGAGAGTG TTAATTCTAG AGAA 13254 INFORMATION FOR SEQ ID NO:157: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157: CAGGTTCAGC TGGTTCAGTC CGGGGCT 27 INFORMATION FOR SEQ ID NO:158: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158: -CCTTGGAGCT CACGATGACC GTGGTTCCTT GGCCCCAGAC GTCC 44 INFORMATION FOR SEQ ID NO:159: SEQUENCE CHARACTERISTICS: LENGTH:.60 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159: GGCCGCGAAT TCGCCGCCAC CATGGAATGG AGCTGGGTCT TTCTCTTCTT CCTGTCAGTA INFORMATION FOR SEQ ID NO:160: 286 SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160: AGCCCCGGAC TGAACCAGCT GAACCTG 27 INFORMATION FOR SEQ ID NO:161: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161: GGAGTTGAGG AGCCTCAGGT CTGCAGACAC GG 32 INFORMATION FOR SEQ ID NO:162: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE.TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162: CCGTGTCTGC AGACCTGTGG CTCCTCAACT CC 32 INFORMATION FOR SEQ ID NO:163: SEQUENCE CHARACTERISTICS: LENGTH: .33 base pairs TYPE: nucleic acid STRANDEDNESS: single 287 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genotnic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163: GATGCCAGAT GTGAGATCGT TCTCACGCAG TCT 33 -INFORMATION FOR SEQ ID NO:164: SEQUENCE CHARACTERISTICS: LENGTH: 67 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164: GCGGGATCCG AATTCTCTAG AATTAACACT CTCCCCTGTT GAAGCTCTTT CTCACCGCC *AACTCAG 67 INFORMATION FOR SEQ ID NO:165:.

SEQUENCE CHARACTE RIST ICS: LENGTH: 51 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoinic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165: GCGCGAATTC ACCATGGGTG TGCCCACTCA GGTCCTGGGG GTTGCTGCTG C 51 INFORMATION FOR SEQ ID NO:166: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic. acid STRANDEDNESS: single TOPOLOGY: linear 288- (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166: ACACTCTC ACAACGATCT CACATCTCCC ATC INFORMATION FOR SEQ ID NO:l67: SEQUENCE CHARACTERISTICS: LENCTH: 50 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genoinic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167: CCCAACCTT ACCATCCCTG TGCCCACTCA CCTCCTCGCG TTCCTCCTC INFORMATION FOR SEQ ID NO:168: SEQUENCE* CHARACTERISTICS: LENGTH: 729 base pairs TYPE:'nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168: GAATTCTCTA CAATTAACAC TCTCCCCTGT TGAACCTCTT TCTCACGCGC CCTGATGGCT GACTTCCCAG CCGTACACTT TGTGTTTCTC CTAGTCTCCT TCAGGCTGCT GCTCACGCTG *TACGTGCT GT CCTTCCTGTC CTCCTCTCTG GGGACTTACC CCATTCGACG CCCTTATCCA 'CCTTC CACTG TACTTTGCC ACAAGTTATT CAGCAGGCAC ACAACAGACG CACTCCCACA TTTCAACTC GCGCGAACAT GAACACACAT GCTGCAGCCA CAGTTCCTTT CCTCTCCAGT GGCCAAAACT CTACCAGCAC GCACCATACA CCTCACAGTA CTACAGTCCA

GAACTCACGC

TTGCTCAGCG

ACACTCTCCT

TCTCTGCGAT

TCATCAGATG

TTGCTCCCCT

AACTCTTCAG

120 180 240 300 360 420 CCTCCACTCT GCTCATCCTC ACAGTGAACT CTGACATGCC ACAGOCCOTA TTCGAAACAC GTTTCTGCTG OTACCAGGCT ACCCGGCGC AGCTCGCTCT TTCCCCTGGA GACAGACACA CA CATCTGGC ATCTGTAAGC CACAGCAGCA

TGGTAACCT

INFORMATION FOR SEQ ID NO:16 SEQUENCE CHARACTERISTIC! LENGTH: 3282 base TYPE: nucleic. acid STRANDEDNESS: daub:.

TOPOLOCY: linear (ii) MOLECULE TYPE: DNA (gen 289- CTGTCCCACA CCCACTCCCG CTCAACCTCT CATGTATGC CACCCTTCCA CCCTCCCCAG TCCGAATCCT CTCACTCCAC CTACACCAGA CGGTCCCTCG ACACTGCCTC

AGAACCATCT

GCAACCCCAG CACCTGAGTC GGCACACCCA 480 540 600 660 720 729 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169: GCATCTACAC ATCATAACAT ACATTCATCA CTTTCCACAA ACCACAACTA CAATCCAGTC

AAAAAAATGC

CTGCAATAAAK

GCTCTGCGAC

TCCCCCTC

CTGTCATCCC

TTTCTCTCAC

CCCGTCCGC

TGCTCTCCCT

TCTCTCCTCC

CTGACTCTCT

CGGACTCCCT

TTTATTTCTG

CAACTTAACA

CTTTTTTAAA

ACGGAAGC

CTGTGTGTCT

CACGTCAGCA

TCCTGCGC

CCTCTCCACT

CCCTGTCGCC

CTC TOCOCAT

TCTCTCCCCA

AAATTTCTCA

ACAACAATTG

GCAAGTAAAA

COCACTOGGA

GTGGCGCCTG

TGTGGAGAAG

CTGTGTCCGA

T CdCCC CCC T

CGCCAACCTC

C TCCACCAGT

TGCGCGTGC

TGCTATTGCT

CATTCATTTT

CCTCTACA.AA

AGTCCCCCAG

CAGCCGCAC

CTGCCCAGAC

GGACTGTTCA

GACCCTC COT

TIATTTGTAA

ATCTTTCACC

TGTGCTATCG

CGCCACCCAC

CTTGTGAGAG

GCTGTCGC

CGTGTCCGAG

CTGTCAGC

CCATTATAAC

TTCAGCGGCA

CTGATTATCA

CTCACCTGCT

GAGGGCTGG

CTTCACCTC

GACCTTGCTC

TCCACCTCTC

CGCCTCTCGA

TCCATCTCCC

120 180 240 300 360 420 480 540 600 660 720 ACTCCGGGGC TTTTCTTGAA CCCTCCACAC GCCCCCACAC ACCCTGGGCA CCACACCCAG CCCTTACTC CGTCCCCCTA CATGCTCAC CGAGCGCCTC TCCCTACGCA CATOCACTAC CCATCCCCC 290 GAGTCGGCAT CGACGTCGGA GGACACGCGG CTCACAGAAC ACGACAGTCT CCTACAGACG

CACGCCTGT

GCTCCTGCTG

GCTGAGCCCC

GTGGGACCCC

AAGAACCATC

GCCG.CCCGCC

CCGGGACCG

TCTCTGTA

GCTCCCACCT

CCACCACGG

CCGCGATCTC

TCAGCTCATC

GGACAGAGGT

CCATCTGCC

GGGCCTGA

TGGTCCACCA

GGCTTTCTCT

GGGTCTTCGT

AGCGTCTCCT

GCTGAGCAG

QGCTACTCTA

TGGGCACGGT

GGGACTTACT

CCTTTGGGGT

CTGTCTGTCC

CAGGCAGGGC

ACCCTAGGCG

CTCTGCCTCC

ACACTCTCC

AAGTATGTAC

GGGCCTTCC

GTCGTTCTC

GCTCTTCTCC

AGCCTGCTC

CCTGCCATAC

CCCCGATGCC

TGGTACAC

CTCCCACCCC

CCGCTTTCTT

CGCTGAGCAC

TCGCATTATG

CCCTCACGTC

TGGCTTTTGC

ACATGCAGCT

GCCCACCTGT

GCCCATGTGT

C CAATCTGGC

GGGCTTAGGT

CCAGGGGCTC

AACGCTGGCA

GCCCAGdGC

CTCATGCCAC

AGCGGCCCAG

AGGGGGTACG

GGCCGTCGCA

AGAGCCTCAT

ACGGTGAGCT

TTGTAGTTGT

AAGCCTTTGA

GGCAGGGTGT

GTCACT.CTCA

ACGGGTCCCA

GGAGACCTTG

GCTGACCACA

CACCTCCA.

CACCACCACG

GGGGAAGAG

GCGTGTCA

CCCGCCTTGA

CAGTTTTGTC

AGGAGAGAAG

CAGGGGGC

CTAGGGCTTC

AAT C C CCCAC ACCTG CACAO

TCAGGCCTCA

OCCAG CCCTG

TGCCAAGCAT

CTCATTTACC

GCATCACGCA

TGCTGTAGAG

TCTCCGCG

CCAGGCACGT

ACACCTGTGC

GGGCAGACGGG

CCTTTCCCTT

CACTTGTACT

CGGTACGTGC

CCTCCACGT

CATGTGACCT

AAGACTGATG

GCACCCCCCT

CCTGGAGGCC

ACAAGATTTG

GTCTCCGAGC

GTCCTCCCCG

CCGTCCCCCA

CCTGCCGAGC

CCTGCCCCAG

GACTCGGCCT

GGTCCTTTAT

CCTCGTGCCA

CGGAGACAGG

GCATGAGAAG

GAAGAACGAG

CCCATTGCTC

CACCCTCACC

TTCTCGGCGC

TCCGCCCAGC

TGGACATGGT

CCTTGCCATT

TCTTGTACTG

ACCAGTTCAA

CAGCGGTCCG

GTCCTCC CC

GCGCCTCTC

CACCCCTCCC

CCCTCTCCAC

TCACCCACTC

ATATCCCTCT

CCCCAGCCCA

GCAGCCCCTC

TGTGCCGACA

CACCCCTGGA

TTCCATCCTG

CCC CACAC

CACACCCTCT

ACCTTCCCCT

CCCTCCCACT

TCCCACTCCA

TCCTTCTTCC

TCCCCTCTAG

CGCCCTCTCT

TTTCTCGCATC

CACCCACTCC

CTCCTCCCC

CTTCACCTCA

CGACATCATC

CCCCTCACCT

OCTGCATGCA

CTCCCTTACC

ACACAACATT

CACACTTTCC

TCCCACCTCT

900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 4$ It 44 4 44 too.

0 CAGCCCAGCA CCTCCCCCTT TCTGTGCAGG CCCTGCCTTA GCCCACCTA CCCTCTCCCT

GCCCAGACC

AGGCCTCCGG

GCCGGGATGC

CTGCTGGCCT

TCCAGATGTA

AGTAGAGTCC

CTCACTTCCA

CCGCTGTGCC

TGGTGGAAGC

TGTCCTGGGG

TGTCTG.CACA

TAAAGGTGAC

TCCATCCCAT

TACTGAATCT

CCTCAGCCCC

TGGGGAAAAA

GTGAAGAGCC

CTCCAGGCAG

CTCACCAACT

GGTCTGCGTG

TGAGGACTGT

CGACACCGTC

CCCAGAGGTG

TGAGCTCACG

AGAATCATCC

CCTGAGCCTC

TCTGTCCTGG

CCACTCAAAC

GTATCCAGAA

GGACTGAACC

GCCAGAAGAC

AGAGCGCCCCC

GACCTCAG

TTCTTGTCCA

CCCAAGCTGC

AGGACACCC

ACCCCTTCGC

CTCTTGGAGG

ATGACCGTG

CAACTATATC

CTCAACTCCA

AACTTCGCTG

CTCTCTCCGG

GCCTGACAAG

AGCTGAACCT

291

CCTCTCCCTG

TGCCTTGCTO

CCTGGCTTCC

CCTTGGTGTT

TGGAGGGCAC

GGAAGGTGTG

GCAAGTAGTC

AGGGTGCCAG

TTCCTTTGCC

CCCCACTCT

TGTAGG CT

AAAATTCTTT

OOOCCTGCC

AAACCTTCAC

GOAGTGGAC

AGCATGAGTG

CCCTGCACTG

AGCAGACAOC

O CTGGGCTTG

GGTCACCACG

CACGCCGCTG

CTTGAGGAGO

GGOGAAGACC

GCAGACGTCC

CCCACAATAA

GTTCGCCAT

GTTTCCGTTG

CACCCAATOA

TGAGGCCCCA

ACCTGTAGTT

GCGGGCAG

GOCCTG dATA

CTCCCTCGCT

TOATTOACOT

CTGCTGACGGG

GTCAGGGCO C GAG CCCAGGG GATGGGCC CT

ATATAATAAT

TAAACAGCCG

GTGTCCGCGG

TAAGGATTGA

ATAACAAAGT

GGCTTCTTCA

ACTGACAGGA

2400 2460 2520 2580 2640 2700 2760 2820 2880 294.0 3000 3060 3120 3180 3240 3282 AGAAGACAAA CACCCACCTC CATICCATOG TCOCGGCGAA TT INFORMATION FOR SEQ ID NO:170: SEQUENCE CHARACTERISTICS: LENGTH: 13254 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170: .TTCTCTACAA TTAACACTCT CCCCTOTTCA AOCTCTTTGT GCGGCAT CTCAOOCCCT GATGOTOAC TTCOCAGCC TACACTTOT OTTTICGA CTCTOCTTTO CTCAOCOTCA GOOTOCTOCT CACOCTOTAG CTGCTOTCCT TGCTGTOCTO CTCTGTOACA CTCTCCTOOO 00 a 00.0 00.

a

AGTTACCCGA

AGTTATTCAG

OGAAGATGAA

GAAAAGTGTA

CCAGTCTGGT

AGATOCCAGA

TOTGCTGGTA

TGGCTCTTTC

ATCTGGGATC

TAAGCTTCGT

GTCTGTCAOC

CTGGCCTCCA

ACAACTCAGC

GTGCAAGCGC

GGACACTG

AAGGAGCTGC

GCTCCGCCGC

ATGCCACGTT

ATTTGTAAAT

AAGGACAGTG

ATTAGTAATG

AGOTATAGOC

AAGCCAAAAA

GCGGTACTTA

CGGAGGCTGG

CAGGCGATCT

TTGGAGGGCG

CAGGCACACA

GACAGATGGT

COAGGAGGCA

GATGGTGAGA

GGCCCTATTG

CCAGGGTACG

CCCTGGAGAC

TOTAAGC GAG

GTCAAGGACG

TATTATGTCT

CCGTTAACAG

TGCCTGATC

GCTCCCCGAG

TGGTGOTGGG

GGAGGGACCA

TAGCGGAGAA

ATOTTTAATA

AATAAATGAG

TATTOO CATA

GAAAGTATCG

TAAGCTATAC

GAGTATAOCA

CGTCACTGTT

ATGGGTC CC C

GACGGTTCAC

TTATGCACCT

AGAGAGOGAG

OCAGO CACAO

CCATAGACOT

GTGAAGTCTG

GAAACACCAT

COG COOCTOC

AGAGACAGGO

AGGAGGAGCA

GTGACGCAG

GGTGGCC

GACGOCAACG

TTCTTCTGCC

CTCATTTTCA

CATCOTGCTG

TGAGT.CGCTG

GAGCCCATGT

AAAAGTGCGG

AGGOCATOCT

TAGCCAATAA

TCAGGAATAG

CATCACCTAT

TGGATAAGAA

GGACOOGGA

GTGTCTTCTA

TAAACGAGCT

292

TCCACTGTAC

TCCCAOATTT

TTCGTTTCCT

GAGAGTAGTA

TCCCAGACCC

OTATOACCAG

OAATO OTOTO

TGCCTOGAGA

ACCCGAGGAC

AAAAGACGCA

GCGGGAGCAA

GGAGTTACCT

GCTGGGTTAA

GACAGATACO

TGCCTAAGCG

GAGGATGGG'

GCGGAACACG

OCACTGOGGA

GTAAAAATAC

ACAOAOTTGT

GCGAOGTG

AACATGAGOA

GCCAAGGGGG

ATCCGGTTC

TGGAGGTCAA

CTGCTTATAT

TTTGOCCTCT

CAAGTGGTCA

GTCCAGTTTG

CAGTGGAAAG

AGTGCGGCTG

GCTTGOAGCC

ACTGGACCTA

CTGCOTGAGA

CTCAGTGGGC

TGCAAAGGAA

CGACTACTOG

CTGACTCTTA

GTCTTCCAAA

CTACCGCCAC

TGGCCTCCAC

AGGAGGOTO

TACCOAGAT

COCTOOTTT

AGACTOCOTO

GGCAAAGAGC

AATAATGGTC

ACOGGOTO

lOGO CCTATA

CAATGCACCG

AACAGGGTGG

AGACCTCCCA

CTGGOATAGA

TCAGATGGCG

OTCCGCTOGC

TOTTOAGOCT

AAGGTGTCTO

TGGCGAGGTT

GAGGAGAAGO

AGGATCTOAG

ACACCOATOG

CGCTTTTA

TCAGACTAGA

TCAGAACAGA

TO COTOAG CO

GOCOTTOTG

TOTTAGGAGC

GGATGTAGAA

TOG COTOGAG

GTATATGTOA

TCAGTCTCTO

CATOTTATOG

AATAAGGCAG

TATAGACCOC

GACTCTATAG

TTCC COGC CO

ATGGOTCTO

CTACAGG

240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 12.60 1320 1380 1440 1500 1560 1620 1680 1740 .<tt S4CZL&C:AS~$t.z 293 S 0 Se..

*5 S S @OS S

S.

S

OS

5 5 55 55..

CTACCGCCCA TTTGCGTCAA TGOGGCGGAG I TTTTGGTGCC AAAACAAACT CCCATTGACG 'I GAGTCAAACC GCTATCGACG CGCATTGATG CGGATOACTA ATACGTAGAT GTACTCCCAA GCATAATGCC AGGCGGGCCA TTTACCGTCA TGATAGACTT GATGTACTGC CAAGTGGGCA CAATGGAAAG TCCCTATTGG CGTTAGTATG CGGGGGTCGT TGGGCGGTCA GCGAGGCGGC TCCATATATG- GGCTATGAAC TAATGACCCC AATCAATCTC AACATGGCGG TAATGTTGGA TATGGATACA ACGTATGCAA TGGCCAATAG AATAATATGG CTAATGGCCA ATATTGATTG CCGCTTGA TdTCGGCTCT ATCGGCGATA AACTGCGATA TTTGCGACAC ACAGAATCGC CGCGTAAACG ATATAAGCGC TATCGCGAGA AAAATCGCGA TATCAGTTAC ACAGAAACTC TTCAAATATC GATTTTTGGA ATATCGCCAT GACATGAATT TACTCGGCGA GAGAAATCTC TATTATTCAC TGCAGTCACC GTCGTTGACA CATTGATGAG TTTGGACAAA CGACAACTAG AATTTGTGAT GCTATTGCTT TATTTGTAAC CAAGA ATTGC ATTCATTTTA TGTTTCAGGT CAAGTAAAAC CTCTACAAAT GTGGTATGGC ATCAAATATT CCTTATTAAG CCCTTTACAA CCATAGTTAT TAATAGCAGA CACTCTATGC TGATTATAAC. TGTTATGCGT ACTTATAAAG TGTTAGGAC ATTTTGGAAA GTCCCGTTGA C~AATGGGGT GGAGACTTGG AAATCCCCGT CACTGCCAAA ACGGCATCAC CATGGTAATA ;TAGGAAAGT CCCATAAGGT CATOTACTGC CTGACGTCAA tAGGGGCGT ACTTGGCATA GTTTACCGTA AATACTCCA CGCATTGACGT GGAACATACG TCATTATTGA CGTCAATGGG CCATTTAGGG TAAGTTATGT AACGCGGAAG GTAATTGATT ACTATTAATA ACTACTCAAT CATCAGCGAA TATAAATGTA CATATTATGA GCAATATTGA TTTATCCTAT ATAACCAATG AATGTATAGA TCGATATCCA TTGGCCATGT TAGCCTCATA TCGTCTGTGA CCTATATCGA CCAAGTGACC AAAGTCGTCT ATCCCCATCC TATCGCGTAT CCCCAAAAAT CACTTTTGGA ACATCGGGGA CATTTTCAAT ATGCCATATT GTCTATCGGC GATAAACACC ACTATCGCGC AAAACGCGTA TTTCG GACAA ACACACATTT CGAAGCTAGA GATCCAGACA TGATAAGATA AATGGAGTGA AAAAAATCCT TTATTTGTGA CATTATAAGC TGCAATAAAC AAGTTAACAA TCACGGGGCAG OTOIGGAGG TTTTTTAAAG TGATTATGAT CTCTAGTCAA GGCACTATAC ATTAAAAAGC TAAAGGTACA CAATTTTTGA GTGTGTGGAG TAAGAAAAAA CACTATGTTA GTTACAGAAT ATTTTTCCAT AATTTTCTTG 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 00 S 006.

TATAGCAGTG

CTAAACACAG

ACCTTTCTCT

CTAGATGGCA

WCCCATTCAT

TTAACACATT

AGTTTGTCCA

CGG GTGGGA

GGAAGGCC

CAAAGGGGTC

CGACAGTCCG

TGATGTTGGA

CATCGTAGGC

TGTCTTCAG

CACCATTCCA

CACATACTCG

ACAGGGTCCT

CTTGACCCCA

TTTGTCTGCG

CCAACCAAAA

GGTGCTGG

TGCAGGCTTC

GAAGGGGTCC

CTGAAAGGTA

ACAGTCGAGG

CATGGCTTGG

CAGCTTTTTC

CATGACTCAA

TGTTTTTTGG

TTTGTTCTGA

CAGTTCCATA

ATACACTTAA

ATTATGTCAC

TGAACTAGGA

GTGGCCAGTC

ACAATTGGCA

GGGAATGCGG

CGTTTCGTGG

TCGAATGTGG

AC CATTCTC C

GTTCCCAGGA

ATCAAGATGA

ATTTGGAGTT

GCATACAAGC

OCCACACCAC

GTGCCCAT

TTCCTCACCA

CGGTTGTACT

CGAAACATCC

CTAGAGCCAT

CTCCGGGTTT

ACTTTCTCAC

CTTTGTGGTG

AAAACTTAGC

AGGAGTAGAA

GCAAAACAG

GGTTGGAATC

AAATTTTATA

ACCACAGAAG

AAGCCTCAAG

TCATTGACAA

GAGGC CC

ATGCTCGCAC

AACCCACTCA

TACCCGTGCC

TCCCCCATGG

ATGGCCTTGG

AACGGGC GAG

CCCACTGGGC

AGCCGCGGTA

AGTAATACGG

CTGTTCC CAT

TGTCCATTAT

TGAAAACTTG

CAACAGGGCT

CAAAATTCCA

TOGAG CGCAG

CCTGGGGGAG

294

TAAATACCAA

AATTCTCAAG

TCTTGAGAGT

TTTTGCTCAT

TAAAATACAC

TTTACCTTAC

TAAGGTTGGT

ATCACTCAAA

GGGATGTGC

CGTCTTGAAA

TGGGATTGGC

GAG CAGGGG

GCTTGGTTAG

CCTTGGTGGT

GGTCAAAGGT

CCAGAGATGA

AGGCATGAC

GTGAGCCTCC

ACCTTGGGGC

CAGAGTATAC

CCGTTTACAC

ACAGAACACC

GAGATACATG

CTCAGGTAAC

TCCTTCTCA

GCACAAGTAC

AGGAAGCAAG

GAAACTCCTT

CAGCACTAGC

TAAAGGCATT

AAACAATTAG

AGCTTTAAAT

TCACAAAGAT

CTCTAATTAG

GACCATGGCT

GTAACCTTTC

GACAC GAG CA

ATTCTCCAGG

TTTCTCGATG

AAAGTTGGTA

TGCTATTACC

TCTCCGATC

TGAGCATTTC

ACGATATCCC

CCAGGAAAGC

TCCTGTTCCA

GAGTGCCTTA

ACCTTGTTGG

TCACTGTTGG

TCTTCTACAC

GTACCATCAA

ATTTGCTTGA

AGTTCTATTA

CGGGTCTTCT

CTGATCATCA

CCACCACTGC

AATCAGTAGT

CTCTGTAGGT

CTCTCTCGCG

TTTTTGTATT

TCTGTCACTG

TTCTCCTGGC

GAAAACTCGT

C CC CCCTTGG

GCCTGCTCGA

TOGGCAGCCTG

CCAAAGTCTT

OOATTCCTTC

TTCCTGTAAT

TGCCATAGGC

CATTCOAACG

TTCCAAACCA

AATTOOTCTC

GATCTCTGCG

AGCCCTCGA

ACTTOOOCTC

CGCAOATATA

TGTTTTTGTT

3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 .4620 4680 4.740 4800 4860 r.

I

1 ±t .00.

so

CAAGTGGGA:A

AGTTTTTGCA

AGO CCGAGGC AATGGCG OA

TTGCTGACTA

GACTAATTGA

CACCCTAAOT

TTTACCAACC

GTOAOCCAGT

AACOATOOGT

TOTCATOATA

AACCCCTATT

ACCCTGATAA

TGTCGCCCTT

GCTGGTGAAA

COATCTCAAC

OAOCACTTTT

GCAACTCGOT

AOAAAAGCAT

OAGTOATAAC

CGCTTTTTTO

GAATGAAGCO

GTTGCGCAAA

CTOGATOGAG

OTTTATTOCT

GGCCAGAT

CTTCCTGAGG

AAAGCCTAGO

GGCCTCGOCC

ACTGOGCGGA

ATTGAOATGC

GATGCATOCT

GACACACATT

TTCTTAAACA

GTGATTACAT

CCCGCAAAGA

ATAATGOTTT

TOTTTATTTT

ATGCTTC.AAT

ATTCCCTTTT

GTAAAAOATG

AGCGOTAAGA

AAAGTTCTGC

.CGCC GGATAC

CTTACCGATG

ACTGCGCCCA

CACAACATG

ATACCAAACG

CTATTAACTC

CCGCATAAAG

CATAAATCTG

C CTAAGCC C

TGGCCATGCT

CCTCCAAAAA

TCTOCATAAA

CTTAGGGG

ATGCTTTGCA

TTGCATACTT

CCACAGCCCC

TAAAGTGTCT

TTTCTCTTCA

CCCATCACCC*

CTTACACGTC

TCTAAATACA

AATATTGAAA

TTC CCCATT

CTOAACATCA

TCCTTCGACAC

TATGGG CGC 295

CCAACGTCCT

ACCCTCCTCA

TAAAAAAAAT

COATGCGCGO

TACTTCTCC

CTGCCTGCTO

ATCTAGCTTG

CCTTATAAAC

CCCTTGTCCT

CCTCGTGATA

ACCIGGCACT

TTOAAATATO

AACOAAGAOT

TTCCCTTCCT

OTTGOICCA

TTTTCGCCCC

CCTATTATCC

CGGCCTO

CTACTTCTCC

TAOTCAGCCA

ACTTAGOGCC

TGCTCGGAC

GGACCCTCC

GCTCCCAAAC

C CACAAAGCC

COCTOCCACG

COCCTATTT

TTTCGGGCAA

TATCCGCTCA

AICACTATIC

GTTTTTCCTC

CCAOTGCGTT

GAACAACCTT

COTOTTGACO

CTTGACTACT

ICGCAGIOCTG

GCAGGACCGA

OATCGTTG

CCTGTAGCAA

TCCCGCCAAC

TCGCCCCTTC

CGCCGTATCA

ACGACCGA

GCCCCAAC CT

AATAOCTCAO

TGCCGCGGAG

COCACTATOG

CCTCCITGC

GGACTTTCOA

CCCCAACCAA

CCACCCGAAG

CAACATTACG

TATAGCTTAA

ATOICCGCOG

TGACACAATA

AACATTTCCG

ACCCAGAAAC

ACATCGAACT

TTCCAATGAT

CCGCCCAACA

CACCAGICAC

CCATAACCAT

ACCAGCTAAC

AACCGGAG CT

TOGCAACAAC

AATTAATAOA

CGC CGOCTG TIC CAGCACT

CTCACCCAAC

4920 4980 5040 5100 5160 5220 5280 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 ACTATICTCA CAATCACTTG OCATOACACT AAGAGjAATTA* ACTTACTTCT GACAACGATC OGGATCATOT AACTCCCCTT ACCAGCCOIA CACCACGATG GCGAACTACT TACTCTAGCT TTCCACCACC ACTTCTCC CAGCCCCCCA GCTGCCTCT CCCGTAICCT AGTTATCTAC £5VSS

TATGCATCAA

ACTGTCACAC

TAAAAGCATC

CTTTTCCTTC

TTTTTTTCTG

TTCTTTGCCC

CCAGATACCA

TGTAGCACCG

CCATAAGTCC

GTCGGGCTGA

ACTOAGATAC

GCACAGGTAT

CCGAAACGCC

ATTTTTGTGA

AGCTCGACGC

GCCCTTCAC

CCCCCCACG

CCAGCCCCAT

CGCCCGTCA

TTGATGAGTT

TTTGTCATGC

ACAATTGCAT

AGTAAAACCT

ACTGCAAGT

GCGGCTCCAG

GAAAGCTG

CGAAATACAC

CAAGTTTACT

TACCTGAAGA

CACTCAGCGT

CGCTAATCT

GATCAACAGC

AATACTGTCC

CCTACATACC

TCTCTTACCG

ACGGGCGTT

CTACACCGTG

CCCCTAAGCG

TGGTATCTTT

TCCTCGTCAG

TCTCCCTTAT

ACCCCCCGC

CCCCTCCCAC

CTTCCCCATC

TCCCCCCAC

TCCACAAACC

TATTGCTTTA

TCATTTTATG

CTACAAATGT

CCCCCACCCC

CCCCACCCTT

CCCACACCCT

ACATCCCTCA

CATATATACT

TCCTTTTTCA

CACACCCCGT

CCTCCTTCCA

TACCAACTCT

TTCTACTCTA

TCCCTCTCCT

CCTTGCACTC

CCTCCACACA

ACCATTCACA

CCACCCTCCC

ATACTCCTCT

CCGCCCCCAC

GCACTCCTC

CAAGGAATCC

CATACCCACC

CCTCATCTCC

CATCCTCCC

ACAACTAGAA

TTTCTAACCA

TTTCACCTTC

CC TATC CC TG

CACCCACCTC

CTCACACGAC

CCTCCCCCTT

296

CATACCTGCC

TTAGATTGAT

TAATCTCATC

ACAAAACATC

AACAAAAAAA

TTTTCCGAAC

GCCCTACTTA

AATCCTCTTA

AACACCATAC

GCCCCACCTTC

AACCCCACC

AACACCACAC

CC CCTTTCCC

CCTATGCAAA

CATTACCAAC

TC CATCCAAC

CCCAAACAAC

GCATATACC

CCTACAGCA

TCCACTCAAA

TTATAACCTG

ACCCCCACCT

ATTATCATCC

ACGTGGTGTC

CC CTCCCTTT

GAGGTCCC

TCACTCATTA

TTAAAACTTC

ACCAAAATCC

AAACCATCTT

CCACCCCTAC

CTAACTCCCT

CGCCACCACT

CCACTCCCTC

TTACCCCATA

CACCAACGA

CTTCCCCAAC

CGCACCACCC

CACCTCTCAC

AACCCCACCA

CACCCCACTA

CACATGCC

CGCTCATCAG

CCCCAGCAAC

TCTACACATC

AAAATCCTTT

CAATAAACAA

CTCGGACCT.T

GCCCTCCACC

T CAT CC CCTC

CTCTCACCAC

CTGCCCCTCC

ACCATTOCTA

ATTTTTAATT

CTTAACCTCA

CTTCACATCC

CACCCTCCT

TCACCACAC

TCAACAACTC

CTGCCAGTCC

AGO CCCACG

CCTACACCGA

CCACAAAGC

ACCTTCCAGC

TTCACTCC

AC CGCC CC

CTACCTTCAC

CCAACACTCC

CCCCAACTCC

CGCACCTCTC

ATAACATACA

ATTTCTGAAA

CTTAACAACA

TTTTAAACCA

CAACCCC

TCTCTCTGTC

CTCACCATCT

TCCCCCCCTC

6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 >2 22 ~2~~>2AA224222~24 ~t2-2S~- TGTCCCACCA CTCTTCACCT CCCGGCTCAG CCTCCCTGTG CAACCTCAGT GCGCGGGTTT CACCAGTCCC TCCACAGCGC.

GGGTCCGAGC CTGCCCACGA GGGCCTCTCC CTACGCACAT CAGGGCGCTC ACACAACACC GGGCCTCCTA GGCCTTCCCG GCTCCCAAAT CCCCCACCCT CACGCACACC TGCACAGCCT ATGCCACTCA GCCCTCAGAC GCCCACCCC ACCCTGCCT GCTACCTGC CAACCATCCT CGTCGCACTC ATTTACCCGC GCCTCATGCA TCACGGAGCA GTGACCTTGC TCTAGAGGAA TAGTTGTTCT CCCGCTCC CCTTTGACCA GCCAGGTCAG AGGGTGTACA CCTCTGGTTC ACTCTCACGG CAGACCGTG GGTCCCA CCT TTGCCTTTGG GACCTTCCAC TTGTACTCCT GACCACACCO TACGTGCTGT CTCCACGCCG TCCACGTACC CACCACGCAT GTCACCTCAG CAAGACGAAG ACTGATGGTC

GTGGAGGAC

TGAGGCGTGC

TGTTGAACGG

CCGAGAGTC

CAGOCAGGG

GGACTAGGCA

ACAGTCTCCT

TGGCC CACCC G CCGAGGGCA

GCCCCAGTGT

TCGGCCTGAC

GCTTTATTTC

CGTGCCACCC

AGACAGGGAG

TGAGAAGACG

GAAGGAGCCG

ATTGCTCTCC

GCTGACCTGG

TCGGGGCTGC

GCCGACCCGG

AGATGGTTTT

TGC CATT GAG

TGTACTGCTC

AGTTGAACTT

GGGTCCGGGA

CTCCCGCGGC

297

CTTGCTCTGG

ACGTGTGTGT

GTGTGGACTG

ATGTCCCGG

TTAGTGCGGT

TGCCCCCGAG

ACAGACGCAG

CAGGGCAGGT

C CC CCTGG CT

GGGGACAGTC

CCGTCGAAAG

CATGCTGGGC

CGAGAGCCCG

AGGCTCTTCT

TTCCCCTGCT

TCGGAGTCCA

CACTCCAG

TTCTTGGTCA

CCTGTAGGCA

CCTCTGTCCA

CTCGATGGGC

CCAGTCCTGG

CTCCCGCGGC

TCTGGGTGCT

GTGGTGGCCG

AGTGTGTGTG

AGTCGGTTGT

GGGGGTAGAT

TGGGCATGGA

GCGCTGTGTG

GCTGCTGGAG

GAGCCGCACC

GGAC CCC CTC

AACCATCACA

GCCCCGGGAAG

GGGACGCG

C CGTGTACTG

GCCACCTGCT

GCACGGCACG

C CAT T CCCT

GCTCATCCCG

CAGAGGTTGG

TGTGGCCCTC

GCTGGGACGC

TGCAGGACGG

TTTGTCTTGG

GTGCAGTTCG

TCTCGCCGGC

TGGGCATCTG

GTCCCCATC

GGGTGAGGCA

GGTCCCACGA

TCTGTCCCCA

GGACGGCAAC

CTAGCGCCC

TCCCTCCCTC

CTCTCCCACC

TAtCTACAGC

CCTTCCC

CTTGTCCACA

CTTCTCCACC

CGTGCTCTTG

GGCATACAAC

GGATCCCCCC

TACACCTC

GCACCCCACC

CTTTGTTCCA.

TCAGCACGCT

CATTATOCAC

TCACCTCCAC

CTTTTCCCCG

TCGACCTGCA

8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940.

9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 CACCTCAGCC .TCTTCGTGC GATCATGACC GTCTCCTTCG CTCACGTCCT GACOGACAGA CGTGTCAGCA CCCGGCTGGG GCCTTGAGCT GGAGGGCGAG TTTTGTCAGA AGATTTGGGC AGAGAAGGTG TCCGAGCTGA CGGCAGGGTC CTCCCGGATA CTGCAGGGCC TGGGTTAGGG AGAAGACCCT CTCCCTGAGC CGGGGCCTGC GTTGCTGCCC COCTOAGOCT GGCTTCCAGC TTGTCGACCT .TGGTGTTGCT AAGCTGCTGG AGGGCACGGT ACAGCCOGGA AGGTGTGGAG CGTTCCCCCA AGTAGTCCTT TTGGAGGAGC GTGGGAGGCC ACCGTGGTTC CTTTGCCCCA CTATATGGCC CCACTCTGGC AACTCCATCT. AGCCCTGTGTT TTGCCTGAA ATTCTTTGTT TGTCCCCCCC CCTGCGCAC TCACAAGAAA CCTTCACTGA TCAACCTGCC :.ACTCGACACC TCCATCGTCC CGGCGAATTC GCAGCCCAAC CTTCGTCTCA CTGTTACTCT GTCAGCTATT ACTACAGTGC CCTGCACCCT AACACAACAA CTCAGCTGCG CCGTGTCC CT

GCCTGGGCTG

TCTC CAGACA

GGGAGTGGAG

TGGCTCTTGG

GCACCTAGC C

ATGAGTCGGGG

TCCACTGCCC

ACACACCCTC

GCCCTTCTGA

CACCACGCTG

GCCGCTGGTC

GACCACGCAC

GAACACCGAT

GACCICCATA

ACAATAATAA

CGCCGATGiG

TCCCTTGTAA

CCAATCAATA

GCCCCAGC

TCTAGTTACT

GAG CTCC CCC

AGGACGCTGA

ATGTCTCCTG

TGCATCTTCT

298

GCATCCAGGC

GCTTACCTGG

GAACATTGGC

ACTTTGGCCT

CAGCTCTGAG

TGTCCCTCC

CGGOAGACO

CTGCATACC

CCTCCCTCTG

TTCACGTTC

CTCACGGAGT

AGOG CCCCTG

CCCAGGGCCG

GGGCCCTTGG

TAATAATTGT

ACAGCCGTGT

TCCGCGGTAA

GGATTGATCC

ACAAAGTTAC

TTCTTCACCT

GACACGAAGA

GGATCGAT

CTCCAGAAAA

CCGCGCGCGG

GCAACGGGAG

TCTGCCGCTG

TACTCTAGGG

GCACGCTGGG

AGTTACTCGA

TTGGGGTGGG

CC GAG GAGCT CACAO CCTGG

CCTCCGGCTG

CGGATCCGTC

CTGGCCTCTC

AGATGTAGGT

AGAGTCCTGA

AGTTCCACGA

CCTTGCC CCC

TGGAAGCTGA

CCTGGGGAGA

-CTGCAGACCT

AGGTGACTCT

ATCCCATCCA

TGAATCTGTA

GAG CCCCGGA

AGAGAAAGAC

CCTCTAGAGT

GACCCATGGA

CAGCAACGAG

TTACCTCTGA

CCTTAAGTCT

GAG CTGTCC

CATGTGTGAG

ATCTGGGACG

CTTAGGTCAG

CCC CCTTTGT

GCAAAAAGCC

AAGAGGCACA

CAGGCACCAG

ACCAACTTTG.

CTGCGTGCCC

GGACTGTAGG

GAG CGTCAC C

AGAGGTGCTC

GCTCACGATG

ATCATCCCAA

GAGGCTCCTC

GTCCTGGAAC

CTCAAACCTC

TGCAGAAGCC

CTGAACCAGC

CGAGCTCCAT

CGATCGACCT

AAGGAACAGT

TACTGCTCAG

CTCTTATCAG

TCCAAATGCG

9600 9660 9720 9780 9840 9900 9960 10020 10080 10140 10200 10260 10320 10380 10440 10500 1 0560 10620 10680.

10740 10800 10860 10920 10980 11040 11100

C.

C. C

C

C

CC C

C*

C

C

TCAGCCGTGC

TTGTGCGGCA

AGGAGCAAGG

GTAGAAGCTC

CGTGGACATCC

ATGTGAATTT

*fCTCTGAAGG

TTATGCATTA

ACCCAGACCT

GACCCCAAGC

CTATAGGCCG

CGGCCGCGGA

CCTCTCCAGG

ACACGCCTAC

CG'rTGATTTT

CCCOTGACT

GTAATAGCGA

.TACTGGGCAT

COCATATGAT

TGACGTCAAT

AATCGCG00

CGGAACTCCA

GTCAATAATC

TTATGATATG

CCAATGAATA

CCATGTGCCA

AAGCCCGCTC

CACTGGTGGT

AGCTGCCGAG

CGCCGCTACC

CACGTTATCT

GTAAATAATA

ACAGTGTATT

GTAATGGAAA

ATAGGCTAAG

CAAAAACAGT

TACTTACGTC

GGCTGGATCG

CGATCTGACG

CGCCCATTTG

GGTGCCAAAA

CAAACCGCTA

TGACTAATAC

AATGCCAGGC

ACACTTGATG

CGAAAGTCCC

CGTCGTTG0G

TATATGGGCT

AATGTCAACA

GATACAACGT

ATATGGCTAA

GCTTGATGTC

CCC0A0CTCA

CGTGGGCATC

CGACCATGAG

GGAGAAGAC

TTAATAAAAA

AATGAGACCC

GGCATATAGC

GTATCGTCAC

CTATACCAIC

ATAGCATGCA

ACTCTTGGCA

GTCCCGGTGT

GTTCACTAAA

CGTCAATGGG

CAAACTCCCA

TCCACGCCCA

GTAGATGTAC

GGGCCATTTA

TACTGCCAAG

TATTGGCGTT

CGGTCAGCCA

ATGAACTAAT

TGGCGGTAAT

ATGCAATGGC

TGGCCAATAT

GCCTCTATG

299-

TTTTCAGACA

GTGCTGTGCC

TCGCTGGAGG

CCATGTCCGG

CTGCGGGCAC

CATCCTGTAA

CAATAAAGAG

CAATAGGA

ACCTATAACA

TAAGAAGCCA

CGGGGAATCC

CTTCTATCCA'

CGAGCTCTGC

GCGGAGTTGT

TTGACGTCAA

TTGATGTACT

TGCCAAGTAG

CCGTCATTGA

TGGGCAGTTT

ACTATGGGAA

GC GC CCAT

GACCCCGTAA

CTTGGACATG

CAATACCCAA

TGATTCAATG

GCGATATAGC

CATACCCTAC

TAAGTCTGGC

CATCGGACCA

AACACCTACC

TGGCCACGCT

AAATACAGAG

AGTTGTGCCA

GTGCTCAATA

TGAGCAAGCG

AGGCGTCGG

GCGTTCCAAT

CGTCAAAACA

TTATATAGAC

TACCACATTT

TCCCGTGCAC

CCCAAAACCG

CAAAGTCCCA

CGTCAATAGC

ACCGTAAATA

CATACGTCAT

TTACCGTAAd

TTCATTACTA

AG CCAATATA

TATTGATTTA

TATAGATCCA

CT CATAT CGT CO COACCO CC

CTCCACTGTT

GGCTCGGAT

CGAGATTOC

CGTGTTGTAT

TCCCTCTCAG

AACAGCCATG

ATGCTCAATA

OCCOTGTATA

CCTATAGACT

GCACCOTTC

GCCTGATOC

CTCC CAC COT

TGOAAAGTCC.

ACTTCGAAAT

CATOACCATG

TAAGGTCATG

CCCCOTACTT

CTCCACCCAT

TATTOACOTC

TTATGTAACC

TTAATAACTA

AATGTACATA

TCCTATATAA

TATGCATTGC

OTGTCACCTA

11160 11220 11280 11340 11400 11460 11520 11580 11640 11700 11760 11820 11880 11940 12000 12060 12120 12180.

12240 12300 12360 1-2420 12480 12540 12600 12660 is S--G-z4

TATCCAAACT

C CATCC C CCG

TTTCCAAAAA

CATATTTTCA

TCGCCCGACA

ACATTTTATT

AACATACATT

TTGTGAAATT

TAACAACAAC

TTAAAGCAAG

CCGATATTTC

TAAACGATAT

TGCCGATATC

AATATCGATT

TGAATTTAGT

ATTCACTCCA

GATGACTTTG

TGTGATCCTA

AATTCCATTC

TAAAACCTCT

CGACACACAG

AAGCCCTATC

ACTTACACAC

TTTCCAATAT

CGGCGACAGA

CTCACCCTCC

CACAAACCAC

TTCCTTTATT

ATTTTATGTT

ACAAATGTGC

300

AATCGCCCAA

CCCAGATATC

AAACTCACAT

CCCCATCTCT

AATCTCAAAA

TTGACACCAA

AACTAGAATG

TCTAACCATT

TCAGCTTCAC

TATGCCTGAT

CTCACCAAAG

CGTATG C CC

CGCCACATT

AT CC CCGATA

CCCTATTTC

CCTACACATC

CACTCAAAAA

ATAACCTCCA

CCCCACCTCT

TAATCATCAA

TCCTCTATCC

AAAAATCACT

TTCAATATC

AACACCACTA

CCACAAACAC

CACACATCAT

AATCCTTTAT

ATAAACAACT

CCCACCTTTT

TCAA

12720 12780 12840 12900 12960 13020 13080 13140 13200 13254

Claims (6)

1. An isolated or substantially purified human whole immunoglobulin molecule which is capable of immunoreacting with human immunodeficiency virus (HIV) glycoprotein and neutralizing HIV, wherein the molecule has the capacity to reduce HIV infectivity titer in an in vitro virus infectivity assay by 50% at a concentration of less than 700 nanograms (ng) of molecule per milliliter (ml) and binds mature gpl20 preferentially over HIV precursor glycoprotein

2. An immunoglobulin molecule according to claim 1, wherein the molecule is IgGl.

3. An immunoglobulin molecule according to claim 1 or claim 2, wherein the molecule has the binding specificity of a monoclonal antibody comprising a heavy chain immunoglobulin variable region amino acid residue sequence shown in SEQ ID NO: 155.

4. A nucleic acid molecule encoding an immunoglobulin according to any one of the preceding claims. e m 25

5. An isolated or substantially purified human whole immunoglobulin molecule which is capable of immunoreacting with HIV glycoprotein gpl20 and neutralising HIV, the molecule being substantially as described herein with reference to the Examples.

6. A nucleic acid molecule encoding the whole immunoglobulin molecule of claim Dated this 30th day of August 2001 THE SCRIPPS RESEARCH INSTITUTE IBy their Patent Attorneys V-0 k/ GRIFFITH HACK