CN111148535A

CN111148535A - Combination therapy for treating solid and hematologic cancers

Info

Publication number: CN111148535A
Application number: CN201880028490.2A
Authority: CN
Inventors: P·T·曼宁; R·普罗; J·C·阿尔马格罗; R·W·卡尔; B·J·卡波恰
Original assignee: Arch Oncology Inc
Current assignee: Arch Oncology Inc
Priority date: 2017-03-22
Filing date: 2018-03-22
Publication date: 2020-05-12
Also published as: SG11201908602UA; AU2018240377A1; EP3600393A4; WO2018175790A1; EP3600393A1; JP7170331B2; JP2020511481A; CA3057139A1

Abstract

Methods are provided for the use of anti-CD 47mAb as a therapeutic agent for the prevention and treatment of solid and hematologic cancers, more specifically chemotherapeutic agents, including, but not limited to, anthracyclines, platins, taxol, topoisomerase inhibitors, antimetabolites, antitumor antibiotics, mitotic inhibitors, and alkylating agents, along with other anticancer agents.

Description

Combination therapy for treating solid and hematologic cancers

Cross Reference to Related Applications

Priority of us provisional application No. 62/475,032 filed on

day

22, 3, 2017, 62/475,036 filed on

day

22, 3, 2017, and us application No. 15/871,802 filed on

day

15, 1, 2018, the contents of which are incorporated herein by reference in their entireties as if written out herein.

Technical Field

The present disclosure relates generally to anti-CD 47 monoclonal antibodies (anti-CD 47 mabs) having different functional profiles as described herein, methods of producing anti-CD 47 mabs, and methods of using these anti-CD 47 mabs in combination with anti-cancer agents as therapeutic agents for the prevention and treatment of solid and hematologic cancers.

Background

CD47 is a cell surface receptor consisting of an extracellular IgV group domain, a 5 transmembrane domain, and an alternatively spliced cytoplasmic tail two ligands bind CD47 Signal inhibitory receptor protein α (SIRP α) and platelet response protein-1 (TSP 1). CD47 expression and/or activity has been implicated in a number of diseases and disorders.

Disclosure of Invention

Compositions and methods for the prevention and treatment of solid and hematologic cancers in combination with anti-cancer agents are provided.

The disclosure describes anti-CD 47 mAbs with different functional profiles, the antibodies have different combinations of properties selected from the group consisting of 1) exhibiting cross-reactivity with one or more species homologs of CD47, 2) blocking the interaction between CD47 and its ligand SIRP α, 3) increasing phagocytosis of human tumor cells, 4) inducing death of susceptible human tumor cells, 5) not inducing cell death of human tumor cells, 6) not having reduced or minimal binding to human red blood cells (hRBC), 7) having reduced binding to hRBC, 8) having minimal binding to hRBC, 9) causing reduced hRBC agglutination, 10) not causing detectable hRBC agglutination, 11) reversing the TSP1 inhibition of the Nitric Oxide (NO) pathway, 12) not reversing TSP1 inhibition of the NO pathway, 13) causing loss of mitochondrial membrane potential, 14) not causing loss of mitochondrial potential, 15) causing increased release of cell surface calponin human tumor cells, 15) not causing increased release of human tumor cells, 10, more specifically NO increase of human endothelial cells, 10, NO human endothelial cells expressing human endothelial cells, NO human endothelial cells expressing NO human endothelial cells, NO human endothelial cells expressing NO human endothelial cells, NO human endothelial cells expressing NO human endothelial cell protein found to increase, NO human endothelial cell line protein found to increase, NO human endothelial cell affinity, NO human endothelial cell line protein found to increase, NO human endothelial cell line (CD 72), NO human endothelial cell line protein found to increase, NO human endothelial cell line expression, NO human endothelial cell line secretion of human endothelial cell line secretion, NO human endothelial cell line secretion of human endothelial cell line secretion, NO human endothelial cell line protein found to human endothelial cell line secretion, NO human endothelial cell line (CD 14) or NO human endothelial cell line (NO) or NO human endothelial cell line secretion of human endothelial cell line (NO) or NO human endothelial cell line secretion of human endothelial cell line (CD 14) or NO human endothelial cell line secretion of human endothelial cell line (CD 14) and NO human endothelial cell line protein found to increase, NO human endothelial cell line (CD 14) or NO human endothelial cell line, NO human endothelial cell line (CD 14) or NO human endothelial cell line secretion of human endothelial cell line, NO human endothelial cell line of human endothelial cell line, NO human endothelial cell line of human endothelial cell line (CD 14) or NO human endothelial cell line, NO human endothelial cell line of human endothelial cell line, NO human endothelial cell.

Embodiments of the disclosure include anti-CD 47 mabs and immunologically active binding fragments thereof; a pharmaceutical composition comprising one or more anti-CD 47mAb, preferably a chimeric or humanized version of said anti-CD 47 mAb; methods of therapeutic use of such anti-CD 47 monoclonal antibodies in combination with an anti-cancer agent.

Embodiments of the disclosure include methods of preventing or treating cancer in a subject by administering to the subject an anti-CD 47 antibody, or antigen-binding fragment thereof, and a second anti-cancer agent.

Embodiments of the disclosure include administering a combination of an anti-CD 47 antibody, or antigen-binding fragment thereof, and a second anti-cancer agent, the administration of which increases tumor cell death compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.

Embodiments of the disclosure include administering a combination of an anti-CD 47 antibody, or antigen-binding fragment thereof, as described herein, and a second anti-cancer agent, the administration of which increases the expression of an immunogenic cell death feature as compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.

Embodiments of the disclosure include administering a combination of an anti-CD 47 antibody and a second anti-cancer agent as described herein, the administration of which increases cell surface calreticulin expression of human tumor cells as compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.

Embodiments of the disclosure include administering a combination of an anti-CD 47 antibody and a second anti-cancer agent as described herein, the administration of which increases ATP release of human tumor cells compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.

Embodiments of the disclosure include a second anticancer agent that is a chemotherapeutic agent.

Embodiments of the present disclosure include chemotherapeutic agents selected from, but not limited to, the class of chemotherapeutic agents consisting of: anthracyclines, platins, taxol, topoisomerase inhibitors, antimetabolites, antitumor antibiotics, mitotic inhibitors, and alkylating agents.

Embodiments of the present disclosure include chemotherapeutic agent classes of anthracyclines selected from, but not limited to, the group consisting of doxorubicin, epirubicin, daunorubicin, and idarubicin.

Embodiments of the disclosure include an anti-CD 47 antibody and a second anticancer agent that is doxorubicin.

Embodiments of the present disclosure include the chemotherapeutic agent class platinum, selected from, but not limited to, the group consisting of oxaliplatin, cisplatin, and carboplatin.

Embodiments of the present disclosure include the chemotherapeutic class of taxol, which is selected from, but not limited to, the group consisting of paclitaxel (paclitaxel) and docetaxel (docetaxel).

Embodiments of the present disclosure include a chemotherapeutic agent class of topoisomerase inhibitors selected from, but not limited to, the group consisting of irinotecan, topotecan, etoposide, and mitoxantrone.

Embodiments of the present disclosure include a chemotherapeutic agent class of antimetabolite, wherein the antimetabolite is selected from the group consisting of 5-FU, capecitabine, cytarabine, gemcitabine, and pemetrexed.

Embodiments of the present disclosure include a chemotherapeutic class of mitotic inhibitors, wherein the mitotic inhibitor is vinorelbine, vinblastine, and vincristine.

Embodiments of the present disclosure include chemotherapeutic class alkylating agents, wherein the alkylating agent is selected from the group consisting of temozolomide.

Embodiments of the present disclosure include an anti-cancer agent, wherein the anti-cancer agent is bortezomib or carfilzomib.

Embodiments of the present disclosure include an anti-CD 47mAb or antigen-binding fragment thereof as defined herein by reference to specific structural features (i.e., the CDRs or specified amino acid sequences of the entire heavy or light chain variable domain). All antibodies of the present disclosure bind CD 47.

A monoclonal antibody or antigen-binding fragment thereof can comprise at least one, typically at least three CDR sequences as provided herein, typically combined with framework sequences from human variable regions or in the form of isolated CDR peptides. In some embodiments, the antibody comprises at least one light chain comprising three light chain CDR sequences provided herein in a variable region framework, which may be, but is not limited to, a murine or human variable region framework, and at least one heavy chain comprising three heavy chain CDR sequences provided herein in a variable region framework, which may be, but is not limited to, a human or murine variable region framework.

Some embodiments of the disclosure are anti-CD 47 mabs or antigen-binding fragments thereof comprising a heavy chain variable domain comprising a variable heavy chain CDR1, a variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 1,2, 3; the variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id no:4, 5, 6; the variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id no: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10.

Heavy chain variable (V)_H) The domain may comprise a combination of any of the listed variable heavy chain CDR1 sequences (HCDR1) with any of the variable heavy chain CDR2 sequences (HCDR2) and any of the variable heavy chain CDR3 sequences (HCDR 3). However, certain embodiments of HCDR 1and HCDR2 and HCDR3 are particularly preferred, derived from a single common V_HDomains, examples of which are described herein.

The antibody or antigen-binding fragment thereof may additionally comprise a light chain variable (V)_L) A domain of a light chain variable domain with V_HThe domains pair to form an antigen binding domain. Preferred light chain variable domains are those comprising a variable light chain CDR1, a variable light chain CDR2, and a variable light chain CDR3, wherein the variable light chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id no:11, 12, 13, 14; the variable light chain CDR2 optionally comprises an amino acid sequence selected from the group consisting of seq id nos: 15, 16, 17; and said variable light chain CDR3 optionally comprises an amino acid sequence selected from the group consisting of seq id nos: 18, 19 and 20.

The light chain variable domain may comprise a combination of any of the listed variable light chain CDR1 sequences (LCDR1) with any of the variable light chain CDR2 sequences (LCDR2) and any of the variable light chain CDR3 sequences (LCDR 3). However, certain embodiments of LCDR 1and LCDR2 and LCDR3 are particularly preferred, and these embodiments are derived from a single common V_LDomains, examples of which are described herein.

Comprises and V_LDomain paired V_HAny of the structural domainsWhat a given CD47 antibody or antigen-binding fragment thereof will comprise a combination of 6 CDRs: variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR1(LCDR 1). While all combinations of 6 CDRs selected from the above listed CDR sequence sets are permissible and within the scope of the disclosure, only certain combinations of 6 CDRs are provided.

Preferred combinations of 6 CDRs include, but are not limited to, combinations of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR3(LCDR3) selected from the group consisting of:

(i) HCDR1 comprising SEQ ID NO. 1, HCDR2 comprising SEQ ID NO. 4, HCDR3 comprising SEQ ID NO. 7, LCDR1 comprising SEQ ID NO. 11, LCDR2 comprising SEQ ID NO. 15, LCDR3 comprising SEQ ID NO. 18;

(ii) HCDR1 comprising SEQ ID NO. 1, HCDR2 comprising SEQ ID NO. 4, HCDR3 comprising SEQ ID NO. 8, LCDR1 comprising SEQ ID NO. 11, LCDR2 comprising SEQ ID NO. 15, LCDR3 comprising SEQ ID NO. 18;

(iii) HCDR1 comprising SEQ ID NO. 2, HCDR2 comprising SEQ ID NO. 5, HCDR3 comprising SEQ ID NO. 9, LCDR1 comprising SEQ ID NO. 12, LCDR2 comprising SEQ ID NO. 16, LCDR3 comprising SEQ ID NO. 19;

(iv) HCDR1 comprising SEQ ID NO. 2, HCDR2 comprising SEQ ID NO. 5, HCDR3 comprising SEQ ID NO. 9, LCDR1 comprising SEQ ID NO. 13, LCDR2 comprising SEQ ID NO. 16, LCDR3 comprising SEQ ID NO. 19; and

(v) HCDR1 comprising SEQ ID NO. 3, HCDR2 comprising SEQ ID NO. 6, HCDR3 comprising SEQ ID NO. 10, LCDR1 comprising SEQ ID NO. 14, LCDR2 comprising SEQ ID NO. 17, LCDR3 comprising SEQ ID NO. 20.

In some embodiments, the anti-CD 47mAb comprises an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of seq id nos: 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of said sequences. Alternatively or additionally, preferred anti-CD 47 mabs (including antibodies or antigen-binding fragments thereof) may comprise a light chain variable domain having an amino acid sequence selected from the group consisting of seq id no:41, 42, 43, 44, 46, 48, 49, 50, 51, and 52, and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of said sequences.

Although selected from the group consisting of V listed above_HAnd V_LV of Domain sequence set_HDomains and V_LAll possible pairings of domains are permissible and within the scope of the disclosure, but V_HAnd V_LCertain combinations of domains are particularly preferred. Thus, a preferred anti-CD 47mAb or antigen-binding fragment thereof is a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) Wherein the combination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 21 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 41;

(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 23 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 43;

(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 49;

(iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 36 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52;

(v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52;

(vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 39 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52;

(vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 24 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 43;

(viii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 37 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52;

(ix) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 33 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(x) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 44;

(xi) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 44; and

(xii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51;

(xiii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 39 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51;

(xiv) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 40 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 52;

(xv) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 36 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51;

(xvi) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 29 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 47;

(xvii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 47;

(xviii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 31 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 47;

(xix) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 32 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 47;

(xx) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 33 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 47;

(xxi) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 29 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxiii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 31 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxiv) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 32 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxv) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 43;

(xxvi) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 43;

(xxvii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 28 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 46;

(xxviii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 35 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 50;

(xxix) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 29 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxx) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 30 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxxi) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 31 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxxii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 32 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(xxxiii) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 37 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51; and

(xxxiv) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 40 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 51.

In some embodiments, the anti-CD 47 antibody or antigen-binding fragment thereof can also comprise a combination of a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises a V having at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98%, or 99% sequence identity to the heavy chain amino acid sequence set forth in (i) to (xxxiv) above_H(ii) a sequence, and/or the light chain variable domain comprises a V having at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity to a light chain amino acid sequence set forth in (i) to (xxxiv) above_LAnd (4) sequencing. Specific V in the sections (i) to (xxxiv)_HAnd V_LPairing or combining V with a particular percentage of sequence identity to these reference sequences_HAnd V_LThe anti-CD 47 antibody of domain sequence may be conserved.

For all embodiments in which the heavy and/or light chain variable domains of an antibody or antigen-binding fragment thereof are defined by a particular percentage of sequence identity to a reference sequence, V_HAnd/or V_LThe domains may retain the same CDR sequences as those present in the reference sequence, such that theThe changes are only present in the framework regions.

In another embodiment, preferred CD47 antibodies or antigen-binding fragments thereof are those comprising a combination of a Heavy Chain (HC) and a Light Chain (LC), wherein the combination is selected from the group consisting of:

(i) a heavy chain comprising the amino acid sequence of SEQ ID NO. 78 and a light chain comprising the amino acid sequence of SEQ ID NO. 67;

(ii) a heavy chain comprising the amino acid sequence of SEQ ID NO. 79 and a light chain comprising the amino acid sequence of SEQ ID NO. 69;

(iii) a heavy chain comprising the amino acid sequence of SEQ ID NO 80 and a light chain comprising the amino acid sequence of SEQ ID NO 70;

(iv) a heavy chain comprising the amino acid sequence of SEQ ID NO 81 and a light chain comprising the amino acid sequence of SEQ ID NO 71;

(v) a heavy chain comprising the amino acid sequence of SEQ ID NO 82 and a light chain comprising the amino acid sequence of SEQ ID NO 71;

(vi) a heavy chain comprising the amino acid sequence of SEQ ID NO 83 and a light chain comprising the amino acid sequence of SEQ ID NO 71;

(vii) a heavy chain comprising the amino acid sequence of SEQ ID NO 84 and a light chain comprising the amino acid sequence of SEQ ID NO 69;

(viii) a heavy chain comprising the amino acid sequence of SEQ ID NO 85 and a light chain comprising the amino acid sequence of SEQ ID NO 71;

(ix) a heavy chain comprising the amino acid sequence of SEQ ID NO 86 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(x) A heavy chain comprising the amino acid sequence of SEQ ID NO 87 and a light chain comprising the amino acid sequence of SEQ ID NO 73;

(xi) A heavy chain comprising the amino acid sequence of SEQ ID NO 88 and a light chain comprising the amino acid sequence of SEQ ID NO 73;

(xii) A heavy chain comprising the amino acid sequence of SEQ ID NO 82 and a light chain comprising the amino acid sequence of SEQ ID NO 74;

(xiii) A heavy chain comprising the amino acid sequence of SEQ ID NO 83 and a light chain comprising the amino acid sequence of SEQ ID NO 74;

(xiv) A heavy chain comprising the amino acid sequence of SEQ ID NO 89 and a light chain comprising the amino acid sequence of SEQ ID NO 71;

(xv) A heavy chain comprising the amino acid sequence of SEQ ID NO 81 and a light chain comprising the amino acid sequence of SEQ ID NO 74;

(xvi) A heavy chain comprising the amino acid sequence of SEQ ID NO. 90 and a light chain comprising the amino acid sequence of SEQ ID NO. 75;

(xvii) A heavy chain comprising the amino acid sequence of SEQ ID NO 91 and a light chain comprising the amino acid sequence of SEQ ID NO 75;

(xviii) A heavy chain comprising the amino acid sequence of SEQ ID NO 92 and a light chain comprising the amino acid sequence of SEQ ID NO 75;

(xix) A heavy chain comprising the amino acid sequence of SEQ ID NO 93 and a light chain comprising the amino acid sequence of SEQ ID NO 75;

(xx) A heavy chain comprising the amino acid sequence of SEQ ID NO 86 and a light chain comprising the amino acid sequence of SEQ ID NO 75;

(xxi) A heavy chain comprising the amino acid sequence of SEQ ID NO 94 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxii) A heavy chain comprising the amino acid sequence of SEQ ID NO 91 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxiii) A heavy chain comprising the amino acid sequence of SEQ ID NO 92 and a light chain comprising the amino acid sequence of SEQ ID NO 31;

(xxiv) A heavy chain comprising the amino acid sequence of SEQ ID NO 93 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxv) A heavy chain comprising the amino acid sequence of SEQ ID NO 87 and a light chain comprising the amino acid sequence of SEQ ID NO 69;

(xxvi) A heavy chain comprising the amino acid sequence of SEQ ID NO 88 and a light chain comprising the amino acid sequence of SEQ ID NO 69;

(xxvii) A heavy chain comprising the amino acid sequence of SEQ ID NO 95 and a light chain comprising the amino acid sequence of SEQ ID NO 76;

(xxviii) A heavy chain comprising the amino acid sequence of SEQ ID NO 96 and a light chain comprising the amino acid sequence of SEQ ID NO 77;

(xxix) A heavy chain comprising the amino acid sequence of SEQ ID NO 97 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxx) A heavy chain comprising the amino acid sequence of SEQ ID NO 98 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxxi) A heavy chain comprising the amino acid sequence of SEQ ID NO 99 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxxii) A heavy chain comprising the amino acid sequence of SEQ ID NO 100 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

(xxxiii) A heavy chain comprising the amino acid sequence of SEQ ID NO 85 and a light chain comprising the amino acid sequence of SEQ ID NO 74;

(xxxiv) A heavy chain comprising the amino acid sequence of SEQ ID NO 89 and a light chain comprising the amino acid sequence of SEQ ID NO 74;

wherein the VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.

In some embodiments, the anti-CD 47 antibodies described herein are also characterized by a combination of properties not exhibited by prior art anti-CD 47 antibodies suggested for human therapeutic use. Accordingly, the anti-CD 47 antibodies described herein are characterized by:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells; and

d. inducing death of susceptible human tumor cells.

In another embodiment described herein, the anti-CD 47 antibody is characterized by:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of susceptible human tumor cells; and

e. no detectable human red blood cell (hRBC) agglutination is caused.

In yet another embodiment described herein, the anti-CD 47 antibody is characterized by:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of susceptible human tumor cells; and

e. causing a reduction in human red blood cell (hRBC) agglutination.

a. specifically binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of susceptible human tumor cells; and

e. with reduced hRBC binding.

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. does not cause detectable human red blood cell (hRBC) agglutination; and

e. with minimal binding to hrbcs.

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. causing detectable human blood red blood cell (hRBC) agglutination; and

e. with reduced hRBC binding.

Additional embodiments of the anti-CD 47 antibodies described herein also feature combinations of properties not exhibited by prior art anti-CD 47 antibodies suggested for human therapeutic use. Thus, the anti-CD 47 antibodies described herein are further characterized by one or more of the following features:

a. causing an increase in cell surface calreticulin expression of human tumor cells;

b. causing an increase in Adenosine Triphosphate (ATP) release from human tumor cells;

c. causing an increase in the release of high mobility group box 1 protein (HMGB1) from human tumor cells;

d. causing an increase in annexin a1 release from human tumor cells;

e. causing an increase in the release of type I interferon from human tumor cells;

f. causes an increase in C-X-C motif chemokine ligand 10(CXCL10) release from human tumor cells;

g. causing an increase in the expression of cell surface protein disulfide isomerase A3(PDIA3) of human tumor cells;

h. causing an increase in the expression of cell surface heat shock protein 70(HSP70) of human tumor cells; and

i. causing an increase in the expression of cell surface heat shock protein 90(HSP90) by human tumor cells.

In another embodiment described herein, the monoclonal antibody or antigen-binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat CD 47.

In yet another embodiment described herein, the monoclonal antibody or antigen-binding fragment thereof also specifically binds to non-human primate CD47, wherein the non-human primate can include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey, and squirrel monkey.

In yet another embodiment described herein, the monoclonal antibody or antigen-binding fragment thereof has reduced binding to normal human cells including, but not limited to, endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripheral blood CD3+ cells, and human peripheral blood mononuclear cells).

In yet another embodiment described herein, the monoclonal antibody or antigen-binding fragment thereof has greater affinity for human CD47 at acidic pH than at physiological pH.

In some embodiments, the monoclonal antibody or antigen binding fragment thereof may additionally have one or more of 1) exhibits cross-reactivity with one or more species homologs of CD47, 2) blocks the interaction between CD47 and its ligand SIRP α, 3) increases phagocytosis of human tumor cells, 4) induces death of susceptible human tumor cells, 5) does not induce cell death of human tumor cells, 6) does not have reduced or minimal binding to human red blood cells (hRBC), 7) has reduced binding to hRBC, 8) has minimal binding to hRBC, 9) causes reduced hRBC, 10) does not cause detectable hRBC agglutination, 11) reverses the Nitric Oxide (NO) pathway TSP1 inhibition, 12) does not reverse NO pathway TSP1 inhibition, 13) causes loss of mitochondrial membrane potential, 14) does not cause loss of mitochondrial membrane potential, 15) causes increased release of human tumor cell surface calsin expression on human tumor cells, 16) does not cause increased release of human reticulocyte (TSP) on human tumor cells, 16) does not cause increased release of human reticulocyte (HSP) and human endothelial cells, 16) does not cause increased release of human tumor cells, 2) of human endothelial cells, 2) does not cause increased endothelial cells, 2, 16, 18, 16, 18, 26, 18, 24, 18, and 24, 18.

Various forms of the disclosed anti-CD 47 mabs are contemplated herein. For example, the anti-CD 47mAb may be a full length humanized antibody having the human framework and constant regions of isotypes IgA, IgD, IgE, IgG, and IgM, more specifically IgG1, IgG2, IgG3, IgG4, and in some cases with different mutations that alter Fc receptor function or prevent Fab arm exchange, or antibody fragments as disclosed herein, e.g., F (ab')2 fragments, F (ab) fragments, single chain Fv fragments (scFv), and the like.

In some embodiments, the therapy provides a combination of an agent that binds CD47 and a second agent that is an anti-cancer agent. Particular combinations of interest include anti-CD 47mAb as disclosed herein and anthracyclines such as doxorubicin, epirubicin, daunorubicin, and idarubicin, which are particularly useful for treating breast cancer, ovarian cancer, gastric cancer, and hepatocellular carcinoma. The combination of anti-CD 47 and a platinum group, such as oxaliplatin, cisplatin, and carboplatin, is particularly useful in the treatment of CRC and NSCLC. The combination of anti-CD 47 and taxol (e.g., paclitaxel and docetaxel) is particularly useful for treating breast cancer, NSCLC, gastric cancer, and prostate cancer. The combination of anti-CD 47 and cyclophosphamide is particularly useful in the treatment of breast cancer. The combination of anti-CD 47 and a topoisomerase inhibitor, e.g., irinotecan, topotecan, etoposide, and mitoxantrone, is particularly useful for treating CRC, small cell lung cancer, pancreatic cancer, ovarian cancer, NSCLC, and NSCLC. The combination of anti-CD 47 and an alkylating agent, such as temozolomide, is particularly useful for treating GBM, melanoma, and multiple myeloma. In some embodiments, providing a therapy that combines agents of CD47 with radiation may also achieve additive or synergistic effects for a variety of solid cancer and hematologic cancer indications.

In some embodiments, pharmaceutical or veterinary compositions are provided comprising an anti-CD 47mAb or one or more of the fragments disclosed herein (optionally in chimeric or humanized form) and a pharmaceutically acceptable carrier, diluent, or excipient.

Some embodiments of the present disclosure provide pharmaceutical compositions comprising one of the anti-CD 47mAb or the fragments disclosed herein (optionally in chimeric or humanized form) in combination with an anti-cancer agent, and a pharmaceutically acceptable carrier, diluent, or excipient.

Prior to the present disclosure, it was necessary to identify anti-CD 47 mabs with a functional profile as described herein. The anti-CD 47 mabs of the present disclosure exhibit different combinations of properties, particularly in combination with anti-cancer agents, making the anti-CD 47mAb particularly advantageous or suitable for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers.

In some embodiments, the disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to human CD47, blocks binding of SIRP α to human CD47, increases phagocytosis of human tumor cells, and induces death of human tumor cells, wherein the monoclonal antibody or antigen-binding fragment thereof exhibits pH-dependent binding to CD47 present on cells, in other embodiments, the disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to human CD47, blocks binding of SIRP α to human CD47, increases phagocytosis to human tumor cells, wherein the monoclonal antibody or antigen-binding fragment thereof exhibits pH-dependent binding to CD47 present on cells, in other embodiments, the disclosure provides a monoclonal antibody or antigen-binding fragment thereof that binds to human CD47, blocks binding of human CD47, increases phagocytosis of human tumor cells, and induces binding of human tumor cells to human peripheral blood cells, wherein the human peripheral blood cells exhibit pH-dependent binding to human PBMC, peripheral blood cells, or human peripheral blood cells, such as human endothelial cells, or peripheral blood cells exhibit increased binding to human endothelial cells, such as human endothelial cells, and normal endothelial cells.

Further areas of applicability of the present disclosure will become apparent from the detailed description provided hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

Drawings

The above and other aspects, features and advantages of the present disclosure will be better understood from the following detailed description taken in conjunction with the accompanying drawings, all of which are given by way of illustration only and are not limiting of the present disclosure.

FIG. 1A.Binding of VLX4 humanized mAb to human OV10 cells expressing human CD 47.Binding of VLX4 humanized mabs (VLX4hum _01 IgG1, VLX4hum _02IgG1, VLX4hum _01IgG4PE, and VLX4hum _02IgG4PE) to human CD47 was determined using ELISA based on OV10 cell line expressing human CD47 (OV10 hCD47) cells. OV10hCD47 cells were seeded into 96-well plates and confluent at the time of assay. Different concentrations of mAb were added to the cells for 1 hour. Cells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

FIG. 1B.Binding of VLX4 humanized mAb to human OV10 cells expressing human CD 47.Binding of VLX4 humanized mabs (VLX4hum _06 IgG4PE, VLX4hum _07 IgG4PE, VLX4hum _12 IgG4PE, and VLX4hum _13 IgG4PE) to human CD47 was determined using an OV10 CD47 cell-based ELISA. OV10hCD47 cells were seeded into 96-well plates and confluent at the time of assay. Different concentrations of VLX4 representative mAb were added to the cells for 1 hour. Cells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

Fig. 2A.Binding of VLX4 humanized mAb to human rbc (hrbc).Binding of VLX4 humanized mabs (VLX4hum _01 IgG1, VLX4hum _02IgG1, VLX4hum _01IgG4PE, and VLX4hum _02IgG4PE) to human CD47 was determined using freshly isolated hrbcs. Hrbcs were incubated with different concentrations of VLX4 mAb for 60 minutes at 37 ℃, washed and incubated with FITC-labeled donkey anti-human antibody for 1 hour. Cells were washed and antibody binding was measured using flow cytometry.

Fig. 2B.Binding of VLX4 humanized mAb to human RBCs.Binding of VLX4 humanized mabs (VLX4hum _07 IgG4PE, VLX4hum _12 IgG4PE, and VLX4hum _13 IgG4PE) to human CD47 was determined using freshly isolated hrbcs. Hrbcs were incubated with different concentrations of VLX4 mAb for 60 minutes at 37 ℃, washed and incubated with FITC-labeled donkey anti-human antibody for 1 hour. Cells were washed and antibody binding was measured using flow cytometry.

Fig. 3A.Binding of the VLX8 humanized mAb to human OV10hCD47 cells.Use baseBinding of human CD47 to VLX 8IgG 4PE chimeric (xi) or humanized mabs (VLX8hum _01IgG4PE, VLX8hum _04 IgG4PE, VLX8hum _07 IgG4PE, and VLX8hum _09 IgG4PE) was determined in an ELISA on OV10hCD47 cells. OV10hCD47 cells were seeded into 96-well plates and confluent at the time of assay. Different concentrations of VLX8 representative mAb were added to the cells for 1 hour. Cells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

Fig. 3B.Binding of the VLX8 humanized mAb to human OV10hCD47 cells.Binding of VLX8 chimeric or humanized mabs (VLX8hum _06IgG2, VLX8hum _07 IgG2, VLX8hum _08IgG2, and VLX8hum _09IgG2) to human CD47 was determined using an OV10hCD47 cell-based ELISA. OV10hCD47 cells were seeded into 96-well plates and confluent at the time of assay. Different concentrations of VLX8 representative mAb were added to the cells for 1 hour. Cells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

Fig. 4A.Binding of VLX8 humanized mAb to human RBCs.Binding of VLX 8IgG 4PE xi or humanized mabs (VLX8hum _01IgG4PE, VLX8hum _03 IgG4PE, VLX8hum _07 IgG4PE, and VLX8hum _10 IgG4PE) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated with different concentrations of VLX8 mAb for 1 hour at 37 ℃, washed and incubated with FITC-labeled donkey anti-human antibody for 1 hour. Cells were washed and antibody binding was measured using flow cytometry.

Fig. 4B.Binding of VLX8 humanized mAb to human RBCs.Binding of VLX 8IgG 4PE xi or humanized mabs (VLX8hum _06IgG2, VLX8hum _07 IgG2, VLX8hum _08IgG2, and VLX8hum _09IgG2) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated with different concentrations of VLX8 mAb for 1 hour at 37 ℃, washed and incubated with FITC-labeled donkey anti-human antibody for 1 hour. Cells were washed and antibody binding was measured using flow cytometry.

Fig. 5A.Binding of the VLX9 humanized mAb to human OV10hCD47 cells.VLX 9IgG2 xi or humanized mAbs (VLX9hum _01 IgG2, VLX9hum _02 IgG2, VLX9hum _03IgG2, V.sub.L) were assayed using an ELISA based on OV10 human CD47 cells,VLX9hum _04IgG2, and VLX9hum _05 IgG2) to human CD 47. OV10hCD47 cells were seeded into 96-well plates and confluent at the time of assay. Different concentrations of mAb were added to the cells for 1 hour. Cells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

Fig. 5B.Binding of the VLX9 humanized mAb to human OV10hCD47 cells.Binding of VLX 9IgG2 xi or humanized mabs (VLX9hum _06IgG2, VLX9hum _07 IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, and VLX9hum _10 IgG2) to human CD47 was determined using an OV10hCD47 cell-based ELISA. OV10hCD47 cells were seeded into 96-well plates and confluent at the time of assay. Different concentrations of mAb were added to the cells for 1 hour. Cells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

Fig. 6A.Specific binding of VLX humanized mAb to CD47Binding of VLX humanized mAbVLX4hum _07 IgG4PE to wild-type and CD47 knock-out Jurkat cells was determined by flow cytometry. Different concentrations of mAb were added to 1X 10⁴In one cell, for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour. Cells were washed and antibody binding was measured using flow cytometry.

Fig. 6B.Specific binding of VLX humanized mAb to CD47Binding of VLX humanized mAbVLX9hum _04IgG2 to wild-type and CD47 knock-out Jurkat cells was determined by flow cytometry. Different concentrations of mAb were added to 1X 10⁴In one cell, for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour. Cells were washed and antibody binding was measured using flow cytometry.

Fig. 7.Binding of VLX9 humanized mAb to human RBCs.Binding of VLX 9IgG2 xi or humanized VLX9 mAb to human CD47(VLX9hum _01 IgG2, VLX9hum _02 IgG2, and VLX9hum _07 IgG2) was determined using freshly isolated human hrbcs. RBCs were incubated with different concentrations of VLX9 mAb for 60 minutes at 37 ℃, washed and incubated with FITC-labeled donkey anti-human antibody for 1 hour. Cells were washed and antibodies were measured using flow cytometryAnd (4) combining.

Fig. 8A.Binding of VLX humanized mAb to Human Aortic Endothelial Cells (HAEC).Binding of VLX humanized mabs (VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11 IgG4PE, VLX4hum _01IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, VLX9hum _03IgG2, and VLX9hum _04IgG2) to HAEC was determined by flow cytometry. HAEC was removed from the flask using agkistrose (acutase). Different concentrations of mAb were added to 1X 10⁴In one cell, for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour, followed by measurement of FITC-label by flow cytometry.

Fig. 8B.Binding of VLX humanized mAb to human skeletal muscle cells (SkMC).Binding of VLX humanized mabs (VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11 IgG4PE, VLX4hum _01IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, VLX9hum _03IgG2, and VLX9hum _04IgG2) to SkMc was determined by flow cytometry. SkMC was removed from the flask using agkistrodon enzyme. Different concentrations of mAb were added to 1X 10⁴In one cell, for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour, followed by measurement of FITC-label by flow cytometry.

Fig. 8C.Binding of the VLX humanized mAb to human pulmonary microvascular endothelial cells (HMVEC-L).Binding of VLX humanized mabs (VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11 IgG4PE, VLX4hum _01IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, VLX9hum _03IgG2, and VLX9hum _04IgG2) to HMVEC-L was determined by flow cytometry. The HMVEC-L was removed from the flask using Agkistrodon enzyme. Different concentrations of mAb were added to 1X 10⁴In one cell, for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour, followed by measurement of FITC-label by flow cytometry.

Fig. 8D.Binding of VLX humanized mAb to human tubular epithelial cells (RTECs).Flow cytometry determination of VLX humanized mAbs (VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11 IgG4PE, VLX4hum _01IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, VLX8hum _11IgG4,VLX9hum _03IgG2 and VLX9hum _04IgG2) to RTEC. RTEC was removed from the flask using agkistrodon enzyme. Different concentrations of mAb were added to 1X 10⁴In one cell, for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour, followed by measurement of FITC-label by flow cytometry.

Fig. 8E. ⁺Binding of VLX humanized mAb to human peripheral blood CD3 cells.Determination of VLX humanized mAbs (VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11 IgG4PE, VLX4hum _01IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, VLX9hum _03IgG2, and VLX9hum _04IgG2) and CD3 by flow cytometry⁺Binding of cells. Will CD3⁺Cells were seeded into 96-well plates. Different concentrations of mAb were added to the cells for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour, followed by measurement of FITC-label by flow cytometry.

Fig. 8F.Binding of VLX humanized mAb to human Peripheral Blood Mononuclear Cells (PBMC).Binding of VLX humanized mabs (VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11 IgG4PE, VLX4hum _01IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09IgG2, VLX9hum _03IgG2, and VLX9hum _04IgG2) to PBMC was determined by flow cytometry. PBMCs were seeded into 96-well plates. Different concentrations of mAb were added to the cells for 1 hour. Cells were washed and then incubated with FITC-labeled secondary antibody for 1 hour, followed by measurement of FITC-label by flow cytometry.

Fig. 9A.pH-dependent and pH-independent binding of humanized mAbs to His-CD 47.Binding of VLX9hum _09IgG2 to human CD47 was determined using a solid phase CD47 ELISA assay. His-CD47 was adsorbed into microtiter wells, washed and various concentrations of humanized mAb were added to the wells at

pH

6 or 8 for 1 hour. The wells were washed and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate.

Fig. 9B.pH-dependent and pH-independent binding of humanized mAbs to His-CD 47.Binding of VLX9hum _04IgG2 to human CD47 was determined using a solid phase CD47 ELISA assay. Adsorbing His-CD47To microtiter wells, wash and add various concentrations of humanized mAb to wells at

pH

Fig. 9C.pH-dependent and pH-independent binding of humanized mAbs to His-CD 47.Binding of VLX4hum _07 IgG4PE to human CD47 was determined using a solid phase CD47 ELISA assay. His-CD47 was adsorbed into microtiter wells, washed and various concentrations of humanized mAb were added to the wells at

pH

Fig. 9D.pH-dependent and pH-independent binding of humanized mAbs to His-CD 47.Binding of VLX8hum _10 IgG4PE to human CD47 was determined using a solid phase CD47 ELISA assay. His-CD47 was adsorbed into microtiter wells, washed and various concentrations of humanized mAb were added to the wells at

pH

Fig. 10.VLX4, VLX8, and VLX9 humanized mabs blocked the binding of SIRP α to CD47 on human Jurkat cells.At 37 ℃, 1.5X 10⁶Individual Jurkat cells were incubated with 5 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _10 IgG4PE, VLX4hum _11 IgG4PE, VLX9hum _03IgG2, VLX9hum _06IgG2, and VLX9hum _08 IgG2) or control antibodies in RPMI containing 10% medium for 30 minutes, an equal volume of fluorescently labeled SIRP α -Fc fusion protein was added and incubated at 37 ℃ for an additional 30 minutes, the cells were washed and binding assessed using flow cytometry.

Fig. 11.The VLX4 CD47 chimeric mAb increases phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml VLX4 chimeric mAb were added to macrophage cultures and incubated for 2 hours at 37 ℃. Removal of non-phagocytosed Jurkat cells and wash macrophage cultures extensively. Macrophages were trypsinized and stained for CD 14. Determination of CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

Fig. 12A.The VLX4 humanized mAb increased phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml antibody were added to the macrophage culture and incubated at 37 ℃ for 2 hours. Unwhaged Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Determination of Total CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

Fig. 12B.The VLX4 humanized mAb increased phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml antibody were added to the macrophage culture and incubated at 37 ℃ for 2 hours. Unwhaged Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Determination of Total CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

Fig. 13A.The VLX8 CD47 chimeric mAb increases phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml VLX8 chimeric mAb were added to macrophage cultures and incubated for 2 hours at 37 ℃. Unwhaged Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Determination of Total CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

FIG. 13B.The VLX8 humanized mAb increased phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml antibody were added to the macrophage culture and incubated at 37 ℃ for 2 hours. Unwhaged Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Determination of Total CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

Fig. 14A.The VLX9CD47 chimeric mAb increases phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml VLX9 chimeric mAb were added to macrophage cultures and incubated at 37 ℃ for two hours. Unwhaged Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Determination of Total CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

Fig. 14B.The VLX9 humanized mAb increased phagocytosis of human Jurkat cells by human macrophages.Human macrophages are scaled up to 1 × 10⁴Individual cells/well concentration were seeded in 96-well plates and allowed to grow adherently for 24 hours. Will be 5X 10⁴Individual CFSE (1 μ M) labeled human Jurkat cells and 1 μ g/ml antibody were added to the macrophage culture and incubated at 37 ℃ for two hours. Unwhaged Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Determination of Total CD14 Using flow cytometry⁺CD14 in a population⁺/CFSE⁺Percentage of cells.

Fig. 15A.Cell death of human Jurkat cells was induced by the soluble VLX4 humanized mAb.At 37 ℃ adding Jurkat cells (1X 10)⁴) Incubation with 1. mu.g/ml of VLX4 humanized mAb (VLX4hum _01 IgG1, VLX4hum _01IgG4PE, VLX4hum _02IgG1, VLX4hum _02IgG4PE) in RPMI medium for 24 hours. Cells were then stained with annexin V and signal detected by flow cytometry. Data were shown to be annexin V positive (annexin V)⁺) Of cells (c).

Fig. 15B.Cell death of human Jurkat cells was induced by the soluble VLX4 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1. mu.g/ml of VLX4 humanized mAb (VLX4hum _01 IgG1, VLX4hum _01IgG4PE, VLX4hum _02IgG1, VLX4hum _02IgG4PE) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD negative (annexin V)⁺/7-AAD^-) Of cells (c).

Fig. 15C.Cell death of human Jurkat cells was induced by the soluble VLX4 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1. mu.g/ml of VLX4 humanized mAb (VLX4hum _01 IgG1, VLX4hum _01IgG4PE, VLX4hum _02IgG1, VLX4hum _02IgG4PE) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) Of cells (c).

Fig. 15D.Cell death of human Jurkat cells was induced by the soluble VLX4 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1 μ g/ml of VLX4 humanized mAb (VLX4hum _06 IgG4PE, VLX4hum _07 IgG4PE, VLX4hum _08 IgG4PE, VLX4hum _11 IgG4PE, VLX4hum _12 IgG4PE, VLX4hum _13 IgG4PE) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive (annexin V)⁺) Of cells (c).

Fig. 15E.Cell death of human Jurkat cells was induced by the soluble VLX4 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1 μ g/ml of VLX4 humanized mAb (VLX4hum _06 IgG4PE, VLX4hum _07 IgG4PE, VLX4hum _08 IgG4PE, VLX4hum _11 IgG4PE, VLX4hum _12 IgG4PE, VLX4hum _13 IgG4PE) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD by flow cytometry. Data were shown to be annexin V positive/7-AAD negative (annexin V)⁺The% of cells of/7-AAD-).

Fig. 15F.Cell death of human Jurkat cells was induced by the soluble VLX4 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1 μ g/ml of VLX4 humanized mAb (VLX4hum _06 IgG4PE, VLX4hum _07 IgG4PE, VLX4hum _08 IgG4PE, VLX4hum _11 IgG4PE, VLX4hum _12 IgG4PE, VLX4hum _13 IgG4PE) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD positive (annexin)⁺/7-AAD⁺) Of cells (c).

Fig. 16A.Cell death of human Jurkat cells was induced by the soluble VLX8 CD47 chimeric mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1. mu.g/ml of VLX8 chimeric mAb (VLX 8IgG 1N 297Q xi and VLX 8IgG 4PE xi) in RPMI medium for 24 hours. Cells were then stained with annexin V and analyzed by flow cytometry. Data presented as annexin V positive (annexin V)⁺) Of cells (c).

Fig. 16B.Cell death of human Jurkat cells was induced by the soluble VLX8 chimeric mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1. mu.g/ml of VLX8 chimeric mAb (VLX 8IgG 1N 297Q xi and VLX 8IgG 4PE xi) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data presented as annexin V positive/7-AAD negative (annexin V)⁺The% of cells of/7-AAD-).

Fig. 16C.Cell death of human Jurkat cells was induced by the soluble VLX8 chimeric mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Chimeric mAbs to VLX8 (VLX 8IgG 1N 297Q xi and VLX 8) at 1. mu.g/ml in RPMI mediumIgG4PE xi) were incubated together for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data presented as annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) Of cells (c).

Fig. 16D.Cell death of human Jurkat cells was induced by the soluble VLX8 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1 μ g/ml of VLX8 humanized mAb (VLX8hum _02IgG4PE, VLX8hum _04 IgG4PE, VLX8hum _07 IgG4PE, and VLX8hum _08 IgG4PE) and chimeric mAb VLX 8IgG 4PE in RPMI medium for 24 hours. Cells were then stained with annexin V and analyzed by flow cytometry. Data presented as annexin V positive (annexin V)⁺) Of cells (c).

Fig. 16E.Cell death of human Jurkat cells was induced by the soluble VLX8 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1 μ g/ml of VLX8 humanized mAb (VLX8hum _02IgG4PE, VLX8hum _04 IgG4PE, VLX8hum _07 IgG4PE, and VLX8hum _08 IgG4PE) and chimeric mAb VLX 8IgG 4PE in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD negative (annexin V)⁺The% of cells of/7-AAD-).

Fig. 16F.Cell death of human Jurkat cells was induced by the soluble VLX8 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubation with 1 μ g/ml of VLX8 humanized mAb (VLX8hum _02IgG4PE, VLX8hum _04 IgG4PE, VLX8hum _07 IgG4PE, and VLX8hum _08 IgG4PE) and chimeric mAb VLX 8IgG 4PE in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) Of cells (c).

Fig. 17A.Cell death of human Jurkat cells was induced by the soluble VLX9 chimeric mAb.At 37 deg.C, 1X 10⁴Jurkat cells were chimeric mAb to VLX9CD47 (VLX 9IgG 1N 297Q xi, VLX 9IgG2 xi and VLX 9IgG2 xi) at 1. mu.g/ml in RPMI mediumVLX 9IgG 4PE xi) were incubated together for 24 hours. Cells were then stained with annexin V and the signal analyzed by flow cytometry. Data were shown to be annexin V positive (annexin V)⁺) Of cells (c).

Fig. 17B.Cell death of human Jurkat cells was induced by the soluble VLX9 chimeric mAb.At 37 deg.C, 1X 10⁴Jurkat cells were incubated with 1. mu.g/ml of VLX9CD47 chimeric mAb (VLX 9IgG 1N 297Q xi, VLX 9IgG2 xi and VLX 9IgG 4PE xi) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD negative (annexin V)⁺The% of cells of/7-AAD-).

FIG. 17C.Cell death of human Jurkat cells was induced by the soluble VLX9 chimeric mAb.At 37 deg.C, 1X 10⁴Jurkat cells were incubated with 1. mu.g/ml of VLX9CD47 chimeric mAb (VLX 9IgG 1N 297Q xi, VLX 9IgG2 xi and VLX 9IgG 4PE xi) in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. Data were shown to be annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) Of cells (c).

Fig. 17D.Cell death of human Jurkat cells was induced by the soluble VLX9 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubated with 1. mu.g/ml of VLX9 humanized mAb (VLX9hum _01 to 10 IgG1) and chimeric mAb VLX 9IgG2 xi in RPMI medium for 24 hours. Cells were then stained with annexin V and signal detected by flow cytometry. VLX 9IgG 2(xi) is a murine/human chimera. Data were shown to be annexin V positive (annexin V)⁺) Of cells (c).

FIG. 17E.Cell death of human Jurkat cells was induced by the soluble VLX9 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubated with 1. mu.g/ml of VLX9 humanized mAb (VLX9hum _01 to 10 IgG1) and chimeric mAb VLX 9IgG2 xi in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. VLX 9IgG 2(xi) is a murine/human chimeraAnd (4) combining the components. Data were shown to be annexin V positive/7-AAD negative (annexin V)⁺The% of cells of/7-AAD-).

FIG. 17F.Cell death of human Jurkat cells was induced by the soluble VLX9 humanized mAb.Jurkat cells (1X 10) were cultured at 37 deg.C⁴) Incubated with 1. mu.g/ml of VLX9 humanized mAb (VLX9hum _01 to 10 IgG1) and chimeric mAb VLX 9IgG2 xi in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD and analyzed by flow cytometry. VLX 9IgG 2(xi) is a murine/human chimera. Data were shown to be annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) Of cells (c).

Fig. 18.Induction of mitochondrial Destruction in human Raji (Raji) cells by soluble VLX4, VLX8 and VLX9 humanized mAbs And (6) polarization.At 37 deg.C, 1X 10⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours. Cells were washed and changes in JC-1 dye fluorescence were assessed using flow cytometry. Data are expressed as% of cells with mitochondrial depolarization.

Fig. 19.Soluble VLX4, VLX8, and VLX9 humanized mAbs cause cell surface calreticulin expression on human Ragi cells The increase in yield.At 37 deg.C, 1X 10⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours in RPMI medium. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive cells.

Fig. 20.Soluble VLX4, VLX8, and VLX9 humanized mabs cause cell surface protein disulfide in human ragged cells Increased expression of the bond isomerase A3(PDIA 3).At 37 deg.C1 × 10 of⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours. Cells were washed and PDIA3 expression was assessed using flow cytometry. Data are expressed as% of cells positive for PDIA 3.

FIG. 21.Soluble VLX4, VLX8, and VLX9 humanized mabs increased cell surface HSP70 expression of human lange cells.At 37 deg.C, 1X 10⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2) in RPMI medium, a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours. Cells were washed and HSP70 expression was assessed using flow cytometry. Data are expressed as% of cells positive for HSP 70.

FIG. 22.Soluble VLX4, VLX8, and VLX9 humanized mabs increased cell surface HSP90 expression of human lange cells.At 37 deg.C, 1X 10⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2) in RPMI medium, a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours. Cells were washed and HSP90 expression was assessed using flow cytometry. Data are expressed as% of cells positive for HSP 90.

FIG. 23.Soluble VLX4, VLX8 and VLX9 humanized mabs increase Adenosine Triphosphate (ATP) release from human lange cells And (4) placing.At 37 deg.C, 1X 10⁵Individual cells/ml of Rakig cells were incubated with 10. mu.g/ml of the humanized mAbs VLX4, VLX8 and VLX9CD47 (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2 and VLX9hum _03 IgG2) in RPMI medium for 24-hour incubation with either a negative IgG control antibody or 1. mu.M mitoxantrone as a positive controlThen (c) is performed. Cell-free supernatants were collected and analyzed using ATP assay kit. Data are expressed as pM ATP in the supernatant.

FIG. 24.Soluble VLX4, VLX8, and VLX9 humanized mabs cause high mobility group 1 proteins of human ragged cells (HMGB1) release.At 37 deg.C, 1X 10⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _03IgG2, VLX9hum _06IgG2, and VLX9hum _08 IgG2), a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours in RPMI medium. Cell-free supernatants were collected and analyzed using HMGB1 immunoassay. Data are expressed as ng/ml of HMGB1 in the supernatant.

FIG. 25.Soluble VLX4, VLX8, and VLX9 humanized mabs increased CXCL10 release from human lange cells.At 37 deg.C, 1X 10⁵Cells/ml of Rakibble cells were incubated with 10. mu.g/ml of VLX4, VLX8, and VLX9CD47 humanized mAbs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _03IgG2, VLX9hum _06IgG2, and VLX9hum _08 IgG2), a negative IgG control antibody, or 1. mu.M mitoxantrone as a positive control for 24 hours in RPMI medium. Cell-free supernatants were collected and analyzed using CXCL10 immunoassay. Data are expressed as pg/ml of CXCL10 in the supernatant.

FIG. 26.Induction of mitochondrial depolarization of human Jurkat cells by soluble VLX4, VLX8, and VLX9 humanized mAbs And (4) transforming.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 10 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1 μ M mitoxantrone as a positive control for 24 hours in RPMI medium. Cells were washed and changes in JC-1 dye fluorescence were assessed using flow cytometry. Data are expressed as% of cells with mitochondrial depolarization.

FIG. 27.Soluble VLX4, VLX8, and VLX9 humanized mabs increase cell surface calreticulin of human Jurkat cells And (4) expressing.At 37At the temperature of 1 multiplied by 10 DEG C⁵Individual cells/ml Jurkat cells were incubated with 10 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1 μ M mitoxantrone as a positive control for 24 hours in RPMI medium. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive cells.

FIG. 28.Soluble VLX4, VLX8, and VLX9 humanized mAbs increase cell surface PDIA3 of human Jurkat cells So as to achieve the purpose.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 10 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1 μ M mitoxantrone as a positive control for 24 hours in RPMI medium. Cells were washed and PDIA3 expression was assessed using flow cytometry. Data are expressed as% of cells positive for PDIA 3.

FIG. 29.Soluble VLX4, VLX8, and VLX9 humanized mAbs increase cell surface HSP70 Table of human Jurkat cells So as to achieve the purpose.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 10 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1 μ M mitoxantrone as a positive control for 24 hours in RPMI medium. Cells were washed and HSP70 expression was assessed using flow cytometry. Data are expressed as% of cells positive for HSP 70.

FIG. 30.Soluble VLX4, VLX8, and VLX9 humanized mAbs increase cell surface HSP90 Table of human Jurkat cells So as to achieve the purpose.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were combined with 10. mu.g/ml of the humanized mAbs VLX4, VLX8 and VLX9CD47 (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2 and VLX9hum _03 IgG2) in RPMI medium, a negative IgG control antibody or as a positive controlmu.M mitoxantrone was incubated for 24 hours. Cells were washed and HSP90 expression was assessed using flow cytometry. Data are expressed as% of cells positive for HSP 90.

FIG. 31.Soluble VLX4, VLX8, and VLX9 humanized mabs increased ATP release from human Jurkat cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 10 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _06IgG2, VLX9hum _08IgG2, and VLX9hum _03 IgG2), a negative IgG control antibody, or 1 μ M mitoxantrone as a positive control for 24 hours in RPMI medium. Cell-free supernatants were collected and analyzed using ATP assay kit. Data are expressed as pM ATP in the supernatant.

FIG. 32.Soluble VLX4, VLX8, and VLX9 humanized mabs increased HMGB1 release from human Jurkat cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 10 μ g/ml of VLX4, VLX8, and VLX9CD47 humanized mabs (VLX9hum _01 IgG2, VLX4hum _07 IgG4PE, VLX8hum _11 IgG4PE, VLX9hum _03IgG2, VLX9hum _06IgG2, and VLX9hum _08 IgG2), a negative IgG control antibody, or 1 μ M mitoxantrone as a positive control for 24 hours in RPMI medium. Cell-free supernatants were collected and analyzed using HMGB1 immunoassay. Data are expressed as ng/ml of HMGB1 in the supernatant.

FIG. 33.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with the chemotherapeutic agent doxorubicin resulted in human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 0.03-10. mu.g/ml VLX4hum _07 IgG4PE alone, 0.3-100nM doxorubicin alone, or 0.03-10. mu.g/ml of a combined dose-responsive matrix of VLX4hum _07 IgG4PE and 0.3-100nM doxorubicin in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 34.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with the chemotherapeutic agent doxorubicin resulted in human Jurkat Synergistic or additive cell death of the cells. 1X 105 cells/ml Jurkat cells were incubated with 0.03-10. mu.g/ml VLX4hum _07 IgG4PE alone, 0.3-100nM doxorubicin alone, or a combination dose-responsive matrix of 0.03-10. mu.g/ml VLX4hum _07 IgG4PE and 0.3-100nM doxorubicin in RPMI medium for 24 hours at 37 ℃. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 35 is a schematic view.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with the chemotherapeutic agent doxorubicin resulted in human Jurkat A synergistic or additive increase in cell surface calreticulin expression of the cell. 1X 105 cells/ml Jurkat cells were incubated with 0.03-10. mu.g/ml VLX4hum _07 IgG4PE alone, 0.3-100nM doxorubicin alone, or a combination dose-responsive matrix of 0.03-10. mu.g/ml VLX4hum _07 IgG4PE and 0.3-100nM doxorubicin in RPMI medium for 24 hours at 37 ℃. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive cells.

FIG. 36.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with the chemotherapeutic agent doxorubicin resulted in human Jurkat Synergistic and/or additive ATP release by the cells. 1X 105 cells/ml Jurkat cells were incubated with 0.03-10. mu.g/ml VLX4hum _07 IgG4PE alone, 0.3-100nM doxorubicin alone, or a combination dose-responsive matrix of 0.03-10. mu.g/ml VLX4hum _07 IgG4PE and 0.3-100nM doxorubicin in RPMI medium for 24 hours at 37 ℃. Cell-free supernatants were collected and analyzed using ATP assay kit. Data are expressed as pM ATP in the supernatant.

Figure 37a hRBC were agglutinated by VLX4 humanized mAb. Hemagglutination was assessed after incubation of hRBCs with different concentrations of humanized VLX4 mAb (25. mu.g/mL-0.4 ng/mL) (VLXhum-01 IgG1, VLX4 hum-01 IgG4 PE). Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA. hRBC were added to a U-bottomed 96-well plate with an equal volume of antibody (75. mu.l) and incubated at 37 ℃ for 3 hours and at 4 ℃ overnight.

Figure 37b agglutination of hrbcs by VLX8 chimeric and humanized mabs. Hemagglutination was assessed after incubation of hRBCs with varying concentrations of humanized VLX4 mAb (25. mu.g/mL-0.4 ng/mL) (VLX8hum _01, _02, _03, _08, _09, _10, _11 IgG4PE) and chimeric mAb VLX 8IgG 4PE xi. Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA. hRBC were added to a U-bottomed 96-well plate with an equal volume of antibody (75. mu.l) and incubated at 37 ℃ for 3 hours and at 4 ℃ overnight.

Fig. 38A.Human RBCs were agglutinated by VLX9 humanized mAb.Hemagglutination was assessed after incubation of human RBCs with different concentrations of VLX 9IgG2 chimeric (xi) and humanized VLX9 mAb (VLX9hum _01 to _06IgG 2). Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to a U-bottomed 96-well plate with an equal volume of antibody (75 μ Ι) and incubated for 3 hours at 37 ℃ and overnight at 4 ℃.

Fig. 38B.Human RBCs were agglutinated by VLX9 humanized mAb.Hemagglutination was assessed after incubation of human RBCs with different concentrations of VLX 9IgG2 chimeric (xi) and humanized VLX9 mAb (VLX9hum _06 to _10 IgG 2). Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to a U-bottomed 96-well plate with an equal volume of antibody (75 μ Ι) and incubated for 3 hours at 37 ℃ and overnight at 4 ℃.

FIG. 39.The VLX4 humanized mAb reduced tumor growth in a ragi xenograft model.Using a catalyst containing 5X 10⁶0.1mL of 30% RPMI/70% Matrigel in Raji tumor cell suspension^TM(BD biosciences (BDbiosciences); Bedford (Mass.) mixtures were subcutaneously inoculated with the flanks of female NSG mice. Five days after inoculation, tumor volumes were measured and would have a size of 31-74mm³Mice with accessible tumor volumes were randomly divided into 8-10 mice/group. VLX4hum _07 or PBS (control) administration was started at this point. Mice were treated by intraperitoneal injection with 5mg/kg antibody 5X/week for 4 weeks. Tumor volume and body weight were recorded twice weekly.

FIG. 40 is a schematic view.The VLX8 humanized mAb reduced tumor growth in a ragi xenograft model.Using a catalyst containing 5X 10⁶0.1mL of 30% RPMI/70% Matrigel in Raji tumor cell suspension^TM(BD bioscience Co. (BDBi)osciences); the mixture of Bedford (Bedford, MA), massachusetts was inoculated subcutaneously into the flanks of female NSG mice. Five days after inoculation, tumor volumes were measured and would have a size of 31-74mm³Mice with accessible tumor volumes were randomly divided into 8-10 mice/group. VLX8hum — 10 or PBS (control) administration was started at this point. Mice were treated by intraperitoneal injection with 5mg/kg antibody 5X/week for 4 weeks. Tumor volume and body weight were recorded twice weekly.

FIG. 41.The VLX9 humanized mAb reduced tumor growth in a ragi xenograft model.Using a catalyst containing 5X 10⁶0.1mL of 30% RPMI/70% Matrigel in Raji tumor cell suspension^TM(BD biosciences (BDbiosciences); Bedford (Mass.) mixtures were subcutaneously inoculated with the flanks of female NSG mice. Five days after inoculation, tumor volumes were measured and would have a size of 31-74mm³Mice with accessible tumor volumes were randomly divided into 8-10 mice/group. VLX9hum _08IgG2 or PBS (control) administration was started at this point. Mice were treated by intraperitoneal injection with 5mg/kg antibody 5X/week for 4 weeks. Tumor volume and body weight were recorded twice weekly.

Fig. 42A.Hemoglobin hydration in blood following administration of humanized VLX9 mAb to cynomolgus monkeys by intravenous infusion And (7) flattening.VLX9hum _08IgG2 or vehicle was administered as an one hour intravenous infusion at a dose of 5mg/kg on day 1and 15mg/kg on day 18. Hemoglobin levels were monitored throughout the study and normalized to control values.

Fig. 42B.RBC levels in blood following administration of humanized VLX9 mAb to cynomolgus monkeys by intravenous infusion.VLX9hum _08IgG2 or vehicle was administered as an one hour intravenous infusion at a dose of 5mg/kg on day 1and 15mg/kg on day 18. RBC levels were monitored throughout the study and normalized to control values.

FIG. 43.Cell death of human OV90 cells was induced by soluble VLX4hum _07 IgG4PE humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 0.03-3. mu.g/ml VLX4hum _07 IgG4PE or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were then plated with annexin V and 7-AADStaining and quantification of annexin positive/7-AAD negative (annexin V +/7-AAD-) cells by flow cytometry.

FIG. 44.Cell death of human OV90 cells was induced by soluble VLX4hum _07 IgG4PE humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 0.03-3. mu.g/ml VLX4hum _07 IgG4PE or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 45.Cell death of human OV90 cells was induced by soluble VLX4hum _07 IgG4PE humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 0.03-3. mu.g/ml VLX4hum _07 IgG4PE or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive cells.

FIG. 46.Cell death of human OV90 cells was induced by the soluble VLX9hum _06IgG2 humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 1-100. mu.g/ml VLX9hum _06IgG2 or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 47.Cell death of human OV90 cells was induced by the soluble VLX9hum _06IgG2 humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 1-100. mu.g/ml VLX9hum _06IgG2 or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 48 is a schematic view.Cell death of human OV90 cells was induced by the soluble VLX9hum _06IgG2 humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells and 1-100. mu.g/ml VLX9hum _06IgG2 in MBCD/199 MediumOr 0.42 μ M doxorubicin for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive cells.

Figure 49 cell death of human OV90 cells was induced by soluble VLX8hum _11 IgG4PE humanized mAb. At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 0.03-3. mu.g/ml VLX8hum _11 IgG4PE or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 50.Cell death of human OV90 cells was induced by soluble VLX8hum _11 IgG4PE humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 0.03-3. mu.g/ml VLX8hum _11 IgG4PE or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 51.Cell death of human OV90 cells was induced by soluble VLX8hum _11 IgG4PE humanized mAb.At 37 deg.C, 1X 10⁵Individual cells/ml OV90 cells were incubated with 0.03-3. mu.g/ml VLX8hum _11 IgG4PE or 0.42. mu.M doxorubicin in MBCD/199 medium for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive cells.

FIG. 52 is a schematic view.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with Doxorubicin elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated with 0.03-1. mu.g/ml VLX4hum _07 IgG4PE alone, 0.005-0.42. mu.M doxorubicin alone, or 0.03-1. mu.g/ml VLX4hum _07 IgG4PE and 0.005-0.42. mu.M doxorubicin in RPMI medium in combination dose-responsive matrix for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 53 is a schematic view.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with Doxorubicin elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated with 0.03-1. mu.g/ml VLX4hum _07 IgG4PE alone, 0.005-0.42. mu.M doxorubicin alone, or 0.03-1. mu.g/ml VLX4hum _07 IgG4PE and 0.005-0.42. mu.M doxorubicin in RPMI medium in combination dose-responsive matrix for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 54 is a schematic view.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with epirubicin elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated with 0.03-1. mu.g/ml VLX4hum _07 IgG4PE alone, 0.005-0.42. mu.M epirubicin alone, or 0.03-1. mu.g/ml VLX4hum _07 IgG4PE and 0.005-0.42. mu.M epirubicin in RPMI medium in combination dose-responsive matrix for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 55.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with Doxorubicin elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated with 0.03-1. mu.g/ml VLX4hum _07 IgG4PE alone, 0.005-0.42. mu.M epirubicin alone, or 0.03-1. mu.g/ml VLX4hum _07 IgG4PE and 0.005-0.42. mu.M epirubicin in RPMI medium in combination dose-responsive matrix for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 56.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with docetaxel to elicit human OV10/315 finesse Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells in RPMI mediumA combined dose-responsive matrix of 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.002-0.135 μ M docetaxel alone, or 0.03-1 μ g/ml VLX4hum _07 IgG4PE and 0.002-0.135 μ M docetaxel was incubated together for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 57.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with epirubicin elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵The individual cells/ml OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.002-0.135 μ M docetaxel alone, or 0.03-1 μ g/ml VLX4hum _07 IgG4PE and 0.002-0.135 μ M docetaxel in RPMI medium in combination dose-responsive matrix for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 58.Combination of the soluble VLX4hum _07 IgG4PE humanized mAb with gemcitabine causes human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated for 24 hours with 0.03-1. mu.g/ml of VLX4 hum-07 IgG4PE alone, 0.003-0.3. mu.M gemcitabine alone, or 0.03-1. mu.g/ml of VLX4 hum-07 IgG4PE and 0.003-0.3. mu.M gemcitabine in RPMI medium in combination dose-responsive matrices. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 59.Combination of the soluble VLX4hum _07 IgG4PE humanized mAb with gemcitabine causes human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated for 24 hours with 0.03-1. mu.g/ml of VLX4 hum-07 IgG4PE alone, 0.003-0.3. mu.M gemcitabine alone, or 0.03-1. mu.g/ml of VLX4 hum-07 IgG4PE and 0.003-0.3. mu.M gemcitabine in RPMI medium in combination dose-responsive matrices. Cells were then plated with annexin V and 7-AADStaining and quantification of annexin positive/7-AAD positive (annexin V +/7-AAD +) cells by flow cytometry.

FIG. 60.Combination of the soluble VLX4hum _07 IgG4PE humanized mAb with gemcitabine causes human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated for 24 hours with 0.03-1. mu.g/ml of VLX4 hum-07 IgG4PE alone, 0.003-0.3. mu.M gemcitabine alone, or 0.03-1. mu.g/ml of VLX4 hum-07 IgG4PE and 0.003-0.3. mu.M gemcitabine in RPMI medium in combination dose-responsive matrices. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive and 7 AAD-cells.

FIG. 61.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with irinotecan elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated for 24 hours with 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.63-51nM irinotecan alone, or a combination dose-responsive matrix of 0.03-1 μ g/ml VLX4hum _07 IgG4PE and 0.63-51nM irinotecan in RPMI medium. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 62.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with irinotecan elicited human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated for 24 hours with 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.63-51nM irinotecan alone, or a combination dose-responsive matrix of 0.03-1 μ g/ml VLX4hum _07 IgG4PE and 0.63-51nM irinotecan in RPMI medium. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 63.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with irinotecan elicited human OV10/315 cells Synergistic or additive cell death.At 37 ℃,1×10⁵Individual cells/ml OV10/315 cells were incubated for 24 hours with 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.63-51nM irinotecan alone, or a combination dose-responsive matrix of 0.03-1 μ g/ml VLX4hum _07 IgG4PE and 0.63-51nM irinotecan in RPMI medium. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive and 7 AAD-cells.

FIG. 64 is a schematic view.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with oxaliplatin induced human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.65-52.8 μ M oxaliplatin alone or a combination dose response matrix of VLX4hum _07 IgG4PE and 0.65-52.8 μ M oxaliplatin alone in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 65.Combination of soluble VLX4hum _07 IgG4PE humanized mAb with oxaliplatin induced human OV10/315 cells Synergistic or additive cell death.At 37 deg.C, 1X 10⁵Individual cells/ml OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07 IgG4PE alone, 0.65-52.8 μ M oxaliplatin alone or a combination dose response matrix of VLX4hum _07 IgG4PE and 0.65-52.8 μ M oxaliplatin alone in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 66.Combination of soluble VLX9hum _06 IgG4PE humanized mAb with chemotherapeutic agent doxorubicin induced human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 1-100. mu.g/ml VLX9hum _06IgG2 alone, 0.005-0.42. mu.M doxorubicin alone, or 1-100. mu.g/ml of a combined dose-responsive matrix of VLX9hum _06IgG2 and 0.005-0.42. mu.M doxorubicin in RPMI medium for 24 hours. Followed by annexin V and7-AAD cells were stained and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 67.Combination of soluble VLX9hum _06 IgG4PE humanized mAb with chemotherapeutic agent doxorubicin induced human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 1-100. mu.g/ml VLX9hum _06IgG2 alone, 0.005-0.42. mu.M doxorubicin alone, or 1-100. mu.g/ml of a combined dose-responsive matrix of VLX9hum _06IgG2 and 0.005-0.42. mu.M doxorubicin in RPMI medium for 24 hours. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 68.Combination of soluble VLX9hum _06 IgG4PE humanized mAb with chemotherapeutic agent doxorubicin induced human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated with 1-100. mu.g/ml VLX9hum _06IgG2 alone, 0.005-0.42. mu.M doxorubicin alone, or 1-100. mu.g/ml of a combined dose-responsive matrix of VLX9hum _06IgG2 and 0.005-0.42. mu.M doxorubicin in RPMI medium for 24 hours. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive and 7 AAD-cells.

FIG. 69.Combination of the soluble VLX8hum _11 IgG4PE humanized mAb with the chemotherapeutic Doxorubicin elicits human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated for 24 hours with 0.03-3 μ g/ml VLX8hum _11 IgG4PE alone, 0.005-0.42 μ M doxorubicin alone, or 0.03-3 μ g/ml VLX8hum _11 IgG4PE and 0.005-0.42 μ M doxorubicin in combination dose-responsive matrix in RPMI medium. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD negative (annexin V +/7-AAD-) cells were quantified by flow cytometry.

FIG. 70.Combination of the soluble VLX8hum _11 IgG4PE humanized mAb with the chemotherapeutic Doxorubicin elicits human Jurkat Synergy of cellsOr accumulating cell death.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated for 24 hours with 0.03-3 μ g/ml VLX8hum _11 IgG4PE alone, 0.005-0.42 μ M doxorubicin alone, or 0.03-3 μ g/ml VLX8hum _11 IgG4PE and 0.005-0.42 μ M doxorubicin in combination dose-responsive matrix in RPMI medium. Cells were then stained with annexin V and 7-AAD, and annexin positive/7-AAD positive (annexin V +/7-AAD +) cells were quantified by flow cytometry.

FIG. 71.Combination of the soluble VLX8hum _11 IgG4PE humanized mAb with the chemotherapeutic Doxorubicin elicits human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated for 24 hours with 0.03-3 μ g/ml VLX8hum _11 IgG4PE alone, 0.005-0.42 μ M doxorubicin alone, or 0.03-3 μ g/ml VLX8hum _11 IgG4PE and 0.005-0.42 μ M doxorubicin in combination dose-responsive matrix in RPMI medium. Cells were washed and calreticulin expression was assessed using flow cytometry. Data are expressed as% of calreticulin positive and 7 AAD-cells.

FIG. 72 is a drawing.Combination of the soluble VLX8hum _11 IgG4PE humanized mAb with the chemotherapeutic Doxorubicin elicits human Jurkat Synergistic or additive cell death of the cells.At 37 deg.C, 1X 10⁵Individual cells/ml Jurkat cells were incubated for 24 hours with 0.03-3 μ g/ml VLX8hum _11 IgG4PE alone, 0.005-0.42 μ M doxorubicin alone, or 0.03-3 μ g/ml VLX8hum _11 IgG4PE and 0.005-0.42 μ M doxorubicin in combination dose-responsive matrix in RPMI medium. Cell-free supernatants were collected and analyzed using HMGB1 ELISA kit. Data are expressed as ng/ml HMGB1 in the supernatant.

FIG. 73.The VLX8 humanized mAb reduced tumor growth in the MDA-MB-231 xenograft model.Using a catalyst containing 2X 10⁷0.2mL of an MDA-MB-231 tumor cell suspension 70% RPMI/30% Matrigel^TM(BD biosciences; Bedford, Mass.) the mixture was inoculated in situ into the mammary fat pad of female NSG mice. Nineteen days after inoculation, tumor volumes were measured and would have a size of 55-179mm³Small accessible tumor volumeThe mice were randomly divided into 10 mice/group. VLXh8um — 10 IgG4PE or PBS (control) administration was started at this time. Mice were treated by intraperitoneal injection with 5mg/kg antibody 5X/week for 5 weeks. Tumor volume and body weight were recorded twice weekly.

Detailed Description

Definition of

Unless defined otherwise, technical and scientific terms used in connection with the present disclosure should have the meaning commonly understood by one of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In general, nomenclature used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide and polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art.

As used herein, the terms "CD 47", "integrin-associated protein (IAP)", "ovarian cancer antigen OA 3", "Rh-related antigen" and "MERG" are synonyms and are used interchangeably.

The term "anti-CD 47 antibody" refers to an antibody of the present disclosure that is intended for use as a therapeutic or diagnostic agent, and thus will typically have the required binding affinity for use as a therapeutic and/or diagnostic agent.

As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active proteins of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically binds" or "immunoreactive" with or against>10^-6). Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, Fab fragments, Fab 'fragments, F (ab')2 fragments, single chain Fv fragments, and single-arm antibodies.

As used herein, the term "monoclonal antibody" as applied to an antibody compound of the invention refers to an antibody that is derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, rather than the method by which it was produced. The mabs of the present disclosure are preferably present in a homogeneous or substantially homogeneous population. The full mAb contains 2 heavy chains and 2 light chains.

An "antibody fragment" refers to a molecule that is not an intact antibody, which comprises a portion of an intact antibody and binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

As disclosed herein, "antibody compound" refers to mabs and antigen-binding fragments thereof. Additional antibody compounds that exhibit similar functional properties according to the present disclosure can be generated by conventional methods. For example, mice can be immunized with human CD47 or fragments thereof, the resulting antibodies can be recovered and purified, and can be evaluated to determine whether they have similar or identical binding and functional properties to the antibody compounds disclosed herein by the methods disclosed in examples 3-16 below. Antigen-binding fragments may also be prepared by conventional methods. Methods for generating and purifying Antibodies and antigen-binding fragments are well known in the art and may be found, for example, in Harlow and Lane (1988) Antibodies, a Laboratory Manual [ Antibodies: a Laboratory Manual, Cold spring Harbor Laboratory Press, Cold spring Harbor, New York, chapters 5-8 and chapter 15.

Monoclonal antibodies encompass antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence in a murine antibody (particularly the murine CDRs), while the chain or the remainder of the chain is identical or homologous to the corresponding sequence in a human antibody. Other embodiments of the disclosure include antigen-binding fragments of the monoclonal antibodies that exhibit similar or identical binding and biological properties to the monoclonal antibodies. Antibodies of the disclosure may comprise kappa or lambda light chain constant regions and heavy chain IgA, IgD, IgE, IgG, or IgM constant regions, including the IgG subclasses IgG1, IgG2, IgG3, and IgG4 and in some cases those with different mutations that alter Fc receptor function.

Monoclonal antibodies containing the presently disclosed murine CDRs can be prepared by any of the various methods known to those skilled in the art, including recombinant DNA methods.

A review of current Methods for Antibody Engineering and improvement can be found, for example, in p.chames editions, (2012) Antibody Engineering: Methods and Protocols [ Antibody Engineering: methods and protocols ], second edition (Methods in Molecular Biology, Vol. 907), Humata Press (HumanaPress), ISBN-10: 1617799734; (2011) Antibody Drug Discovery (Molecular Medicine and Medicinal Chemistry, Book 4) [ Antibody Drug Discovery (Molecular drugs and Medicinal Chemistry) ], Imperial College Press (Imperial College Press); editors of r.kontermann and s.dubel, (2010) Antibody Engineering Volumes 1and 2(Springer Protocols) [ Antibody Engineering Volumes 1and 2 (schpringer protocol) ], second edition; and w.strohl and l.strohl (2012) therapeutic engineering Current and future advances driving the string growth area in the pharmaceutical industry [ therapeutic antibody engineering: driving current and future advances in the most intense growing areas in the pharmaceutical industry ], wood sea press (Woodhead Publishing).

Methods for generating and purifying Antibodies and antigen-binding fragments are well known in the art and may be found, for example, in Harlow and Lane (1988) Antibodies, a Laboratory Manual [ Antibodies: a laboratory manual ], cold spring harbor laboratory Press, cold spring harbor, New York, chapters 5-8 and chapter 15.

When present, full-length antibodies are naturally "Y" -type immunoglobulin (Ig) molecules comprising four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains interconnected by disulfide bonds. The amino-terminal portion of each chain, referred to as the fragment antigen-binding region (FAB), includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition via the Complementarity Determining Regions (CDRs) contained therein. The carboxy-terminal portion of each chain defines a constant region ("Fc" region), primarily responsible for effector function.

These CDRs are interspersed with more conserved regions, called frameworks ("FRs"). The amino acid sequences of many FRs are well known in the art. Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRL, FR2, CDR2, FR3, CDR3 and FR 4. The 3 CDRs of the light chain are referred to as "LCDR 1, LCDR2, and LCDR 3" and the 3 CDRs of the heavy chain are referred to as "HCDR 1, HCDR2, and HCDR 3". These CDRs contain most of the residues that form the specific interactions with the antigen. The numbering and positioning of CDR amino acid residues within the LCVR and HCVR regions follows well-known Kabat numbering rules (Kabat et al, (1991) Sequences of Proteins of immunological Interest [ protein Sequences of immunological Interest ], 15 th edition NIH publication No. 91-3242).

As used herein, "antigen binding site" may also be defined as a "hypervariable region", "HVR" or "HV", and refers to the structural hypervariable region of an antibody variable domain, as defined by Chothia and Lesk (Chothia and Lesk, mol. biol. [ molecular biology ]196: 901. 917, 1987). There are six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). CDRs other than H-CDR1 are used herein, which extend to include H1, as defined by Kabat.

There are five types of mammalian immunoglobulin (Ig) heavy chains, represented by the greek letters α (alpha), δ (delta), ε (epsilonlon), γ (gamma), and μ (spurious), which define the class or isotype of an antibody as IgA, IgD, IgE, IgG, or igm, respectively, IgG antibodies can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG 4.

Heavy chains γ, α, and δ have a constant region consisting of three tandem immunoglobulin (Ig) domains and a hinge region for added flexibility.

The hinge region is a flexible amino acid fragment that links the Fc portion and the Fab portion of the antibody. This region contains cysteine residues that can form disulfide bonds linking the two heavy chains together.

The variable region of the heavy chain is different in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or individual B cells. The variable region of each heavy chain is about 110 amino acids in length and consists of a single Ig domain.

In mammals, light chains are classified as kappa (κ) or lanboda (λ), and are characterized by specific constant regions known in the art. The light chain has two contiguous domains: a variable domain at the amino terminus, and a constant domain at the carboxy terminus. Each antibody contains two light chains that are identical at all times; only one type of light chain, κ or λ, is present per antibody in mammals.

The Fc region, consisting of two heavy chains constituting three or four constant domains depending on the antibody class, plays a role in regulating the activity of immune cells. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response to a given antigen. The Fc region also binds to different cellular receptors such as Fc receptors and other immune molecules such as complement proteins. By doing so, it mediates various physiological effects including opsonization, cell lysis, and degranulation of mast cells, basophils, and eosinophils.

As used herein, the term "epitope" refers to the specific arrangement of amino acids on a peptide or protein to which an antibody or antibody fragment binds. Epitopes are usually composed of chemically active surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be linear, i.e. involving binding to a single sequence of amino acids, or may be conformational, i.e. involving binding to two or more sequences of amino acids in different regions of the antigen which may not necessarily be adjacent to a linear sequence.

As used herein, the terms "specifically binds," "specifically binds," and the like, as applied to an antibody compound of the invention, refer to the ability of a specific binding agent (e.g., an antibody) to preferentially bind to a target molecular species as compared to other molecular species, thereby mixing the specific binding agent with the target molecular species. A specific binding agent is considered to specifically recognize a target when it is capable of specifically binding to a target molecule species.

As used herein, the term "binding affinity" refers to the strength with which a molecule binds to another molecule at a site on the molecule. Two molecules are considered to exhibit binding affinity for each other if a particular molecule is to bind to or specifically associate with another particular molecule. Binding affinity is related to the association and dissociation constants of a pair of molecules, but this is not critical to the methods herein, as these constants can be measured or determined. In contrast, affinity as used herein to describe interactions between molecules in these described methods is typically the apparent affinity observed in empirical studies (unless otherwise specified), which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will thereby bind to two other molecules (e.g., two forms or variants of a peptide). The concepts of binding affinity, association constant, and dissociation constant are well known.

As used herein, the term "sequence identity" refers to the percentage of identical nucleotides or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned such that the sequence matches are maximized (i.e., gaps and insertions are considered). Identity can be readily calculated by known methods including, but not limited to, those described in: computational Molecular Biology [ Computational Molecular Biology ], Lesk, a.m. editors, oxford university Press (oxford university Press), new york, 1988; biocontrol information and genome Projects [ biological: informatics and genomics projects ], Smith, d.w. editors, Academic Press, new york, 1993; computer Analysis of Sequence Data, Part I [ Computer Analysis of Sequence Data, Part I ], Griffin, A.M. and Griffin, H.G. eds, Humana Press (Lemama Press), New Jersey, 1994; sequence Analysis in Molecular Biology [ Sequence Analysis in Molecular Biology ], von heinje, g., Academic Press (Academic Press), 1987; and Sequence Analysis Primer [ Sequence Analysis Primer ], Gribskov, M. and Devereux, j. editions, M Stockton Press (stokes Press), new york, 1991; and Carillo, h, and Lipman, d., sia j. applied Math [ journal of applied mathematics of the institute of industrial and applied mathematics ],48:1073 (1988). The method for determining identity is designed to give the largest match between the tested sequences. Furthermore, the method of determining identity is to be hacked in a publicly available computer program.

For example, optimal sequence alignments for comparison can be made by the local homology algorithms of Smith and Waterman, by homology alignment algorithms, by similarity search methods, or by computerized execution of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package (WisconsinPackage), available from Arsennix corporation (Accelrys, Inc.), San Diego, Calif., USA), or by visual inspection. See generally Altschul, S.F. et al, J.mol.biol. [ J.M. J.biol. ]215: 403-.

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul, s. et al, NCBI NLM NIH maryland bessel 20894; and Altschul, S. et al, J.mol.biol. [ J.Mol.M. 215: 403-. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short characters of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is called the neighbor score threshold.

These initial neighborhood character hits serve as the basis for initiating searches to find longer HSPs containing them. Character hits are then extended along each sequence in two directions, provided that the cumulative alignment score can be increased. Cumulative scores were calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues; always; 0) and N (penalty score for mismatching residues; always; 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Stopping the character hit extension in each direction when: the cumulative alignment score is reduced by the amount X from its maximum achieved, the cumulative score is reduced to 0 or below due to the accumulation of one or more negative-scoring residue alignments, or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation (E) of 10, a cutoff (cutoff) of 100, M-5, N-4, and a comparison of the two strands as defaults. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix as defaults.

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences will occur by chance. For example, a test nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1 in one embodiment, less than about 0.01 in another embodiment, and less than about 0.001 in yet another embodiment.

As used herein, the terms "humanized", "humanizing" and the like refer to the CDR grafting of a murine monoclonal antibody as disclosed herein to human FRs and constant regions. These terms also encompass possible further modifications of the murine CDRs and human FRs by methods such as those disclosed separately in the following documents: kashmiri et al (2005) Methods [ Methods ]36(1):25-34 and Hou et al (2008) J. biochem. [ J. Biochem ]144(1): 115-120) to improve different antibody properties, as discussed below.

As used herein, the term "humanized antibody" refers to mabs and antigen-binding fragments thereof, including the antibody compounds disclosed herein, that have binding and functional properties according to the present disclosure similar to those disclosed herein, and have substantially human or fully human FR and constant regions around CDRs derived from non-human antibodies.

As used herein, the term "FR" or "framework sequence" refers to any of FR1 to 4. Humanized antibodies and antigen-binding fragments encompassed by the present disclosure include molecules in which any one or more of FR1 through FR4 is substantially or completely human, i.e., in which any one of the possible combinations of substantially or completely human FR1 through FR4 alone is present. For example, this includes FR 1and FR 2; FR 1and FR 3; FR1, FR2, FR3 and the like are substantially or entirely human molecules. Substantially human framework regions are those having at least 80% sequence identity to known human germline framework sequences. Preferably, the substantially human framework region has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a framework sequence disclosed herein, or to a known human germline framework sequence.

Fully human framework regions are those that are identical to known human germline framework sequences. Human FR germline sequences can be obtained from the International ImmunoGeneTiCs (IMGT) database and Marie-Paule Lefranc and Gerard Lefranc, the Immunoglobin fatsBook, academic Press, 2001, the contents of which are incorporated herein by reference in their entirety.

The immunoglobulin plexus is a summary of human germline immunoglobulin genes used to create human antibody repertoires and includes entries of 203 genes and 459 alleles for a total of 837 sequences exhibited. Individual entries include all human immunoglobulin constant genes and germline variable genes, diversity genes and linking genes with at least one functional or open reading frame allele and located in three major loci. For example, the germline light chain FR may be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39, IGKV1D-33 and IGKJ1-5, and the heavy chain germline FR may be selected from the group consisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20, IGHV3-66, IGHV3-72, IGHV3-74, IGHV4-31, IGHV3-21, IGHV3-23, IGHV3-30, IGHV3-48, IGHV4-39, IGHV4-59, and IGHV5-51, and IGHJ 1-6.

Substantially human FRs are those that have at least 80% sequence identity to known human germline FR sequences. Preferably, the substantially human framework region has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a framework sequence disclosed herein, or to a known human germline framework sequence.

CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the CDR sequences disclosed herein. Alternatively, CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1,2, 3, 4, or 5 amino acid changes at corresponding positions as compared to the CDR sequences disclosed herein. Such a sequence-identical, or amino acid-modified CDR preferably binds to an antigen recognized by an intact antibody.

Humanization began with chimerization, a method developed in the first half of the 80's of the 20 th century (Morrison, s.l., m.j.johnson, l.a.herzenberg, and v.t.oi: nucleic human antibody molecules: human antibody-binding domains with human constant region domains [ Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains ] proc.natl.acad.sci.usa [ national academy of sciences of america ],81,6851-5(1984)), consisting of: comprising combining the variable (V) domain of a murine antibody with a human constant (C) domain to produce a molecule having about 70% human content.

The present disclosure includes humanized antibodies that can be produced using several different methods, including Almagro et al Humanization of antibodies, [ Humanization of antibodies ] Frontiers in Biosciences, [ frontier of Biosciences ] (2008)1 month 1; 13: 1619-33.

In one method, the CDRs of a parent antibody compound are grafted onto a human framework having a high degree of sequence identity to the framework of the parent antibody compound. The sequence identity of the novel framework will generally be at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of the corresponding framework in the parent antibody compound. In the case of a framework having less than 100 amino acid residues, one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues may be changed. Such transplantation may result in a reduction in binding affinity compared to the parent antibody. If so, the framework can be back mutated at certain positions to the parental framework based on specific criteria disclosed by Queen et al (1991) Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. ]88: 2869. Additional references describing methods useful for generating humanized variants based on homology and back-mutation include those described below: biolnformatics, Olimpieri et al [ Bioinformatics ]2015, 2 months, 1 days; 31(3) 434-; and the method of Winter and coworkers (Jones et al (1986) Nature [ Nature ]321: 522-525; Riechmann et al (1988) Nature [ Nature ]332: 323-327; and Verhoeyen et al (1988) Science [ Science ]239: 1534-1536).

The identification of residues for which back-mutations are considered can be performed as follows. The framework amino acids of the human germline sequence being used ("acceptor FR") are replaced by framework amino acids from the framework of the parent antibody compound ("donor FR") when the amino acids fall into the following categories:

(a) the amino acid in the human FR of the acceptor framework is unusual for the human framework at this position, whereas the corresponding amino acid in the donor immunoglobulin is typical for the human framework at this position;

(b) the amino acid is positioned immediately adjacent to one of these CDRs; or

(c) Any side chain atom of a framework amino acid is within about 5-6 angstroms (center-to-center) of any atom of a CDR amino acid of the three-dimensional immunoglobulin model.

When each amino acid in the human FR of the acceptor framework and the corresponding amino acid in the donor framework are collectively rare for the human framework at that position, such an amino group can be replaced by an amino acid that is typical for the human framework at that position. Such back-mutation criteria enable one to restore the activity of the parent antibody compound.

Another method of generating humanized antibodies that exhibit similar functional properties to the antibody compounds of the disclosure involves randomly mutating amino acids within the grafted CDRs without altering the framework, and screening the resulting molecules for binding affinity and other functional properties as good or better than the parent antibody compounds. It is also possible to introduce a single mutation at each amino acid position within each CDR and then evaluate the effect of such mutations on binding affinity and other functional properties. The individual mutations that give rise to the improved properties can be combined in order to assess their effect in combination with one another.

Furthermore, a combination of the two aforementioned methods is possible. After CDR grafting, specific FRs may be back-mutated, in addition to introducing amino acid changes in these CDRs. This method is described in Wu et al (1999) J.mol.biol. [ journal of molecular biology ]294: 151-162.

Using the teachings of the present disclosure, one skilled in the art can use common techniques, such as site-directed mutagenesis, to substitute amino acids within the presently disclosed CDR and FR sequences and thereby generate variable region amino acid sequences derived from the present sequences. Up to all naturally occurring amino acids may be introduced at a particular substitution position. The methods disclosed herein can then be used to screen the additional variable region amino acid sequences to identify sequences with the indicated in vivo function. In this manner, other sequences suitable for preparing humanized antibodies and antigen-binding fragments thereof according to the present disclosure can be identified. Preferably, amino acid substitutions within the framework are limited to one, two, three, four, or five positions within any one or more of the four light chain and/or heavy chain FRs disclosed herein. Preferably, the amino acid substitutions within the CDRs are limited to one, two, three, four, or five positions within any one or more of the three light and/or heavy chain CDRs. Combinations of variations within these FR and CDR's described above are also possible.

The functional properties of the antibody compounds generated by the introduction of the amino acid modifications discussed above, consistent with the functional properties exhibited by the particular molecules disclosed herein, can be demonstrated by the methods in the examples disclosed herein.

As described above, to avoid the problem of eliciting a human anti-mouse antibody (HAMA) response in patients, murine antibodies have been genetically manipulated to become light (V) by grafting their Complementarity Determining Regions (CDRs) to humans_L) Chains and variable weights (V)_H) Those murine framework residues on the chain framework that are believed to be essential for the integrity of the antigen binding site are simultaneously retained to gradually replace their murine content with amino acid residues present in their human counterparts. However, the xenografted CDRs of the humanized antibody can elicit an anti-idiotypic (anti-Id) response in the patient.

To minimize the anti-Id response, procedures have been developed to humanize xenograft antibodies by grafting only the most important CDR residues in antibody-ligand interactions onto the human framework (referred to as "SDR grafting"), where only the important Specificity Determining Residues (SDRs) of the CDRs are grafted onto the human framework. This procedure, described by Kashmiri et al (2005) Methods [ Methods ]36(1):25-34, involves the identification of SDRs either with the aid of a database of the three-dimensional structures of antigen-antibody complexes of known structure or by mutation analysis of the antibody binding sites. An alternative humanization approach involving the retention of more CDR residues is based on the grafting of CDR residue fragments comprising all SDRs, i.e., "shortened" CDRs. Kashmiri et al also disclose a procedure for assessing the reactivity of humanized antibodies with serum from patients to whom murine antibodies have been administered.

Hou et al (2008) J. biochem. [ J. Biochem ]144(1):115-120 disclose another strategy for constructing human antibody variants with improved immunogenic properties. These authors developed humanized antibodies from the murine anti-human CD34 monoclonal antibody 4C8 by CDR grafting using a 4C8 molecular model established by computer-assisted homology modeling. Using this molecular model, these authors identified FR residues of potential importance in antigen binding. Humanized versions of 4C8 were generated by transferring these critical murine FR residues along with the murine CDR residues onto a human antibody framework selected based on homology to the murine antibody FRs. The resulting humanized antibody has been shown to have similar antigen binding affinity and specificity as the initial murine antibody, suggesting that it may be an alternative to the murine anti-CD 34 antibody that is routinely used clinically.

Embodiments of the disclosure encompass antibodies created to avoid recognition by the human immune system that contain CDRs described herein in any combination such that contemplated mabs can contain a set of CDRs from a single murine mAb disclosed herein, or light and heavy chains that contain a set of CDRs that are derived from individual CDRs of two or three disclosed murine mabs. Such mabs can be created by standard techniques of molecular biology and screened for their desired activity using the assays described herein. In this way, the present disclosure provides a "mixed-pair" method of creating novel mabs comprising a mixture of CDRs from the disclosed murine mabs to achieve new or improved therapeutic activity.

Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that "compete" with the molecules disclosed herein are those that bind human CD47 at one or more sites that are the same as or overlap with the one or more sites to which the molecules of the present invention bind. For example, a competing monoclonal antibody or antigen-binding fragment thereof can be identified by an antibody competition assay. For example, a sample of purified or partially purified extracellular domain of human CD47 can be bound to a solid support. Then, an antibody compound of the disclosure, or an antigen-binding fragment thereof, and a monoclonal antibody or an antigen-binding fragment thereof suspected of being able to compete with the disclosed antibody compound are added. One of these two molecules is labeled. If the labeled compound and the unlabeled compound bind to separate and discrete sites on CD47, the labeled compound will bind to the same level regardless of whether the suspected competitive compound is present. However, if these sites of interaction are identical or overlap, the unlabeled compound will compete and the amount of labeled compound bound to the antigen will be reduced. If the unlabeled compound is present in excess, little, if any, of the labeled compound will bind. For purposes of this disclosure, competitive monoclonal antibodies, or antigen-binding fragments thereof, are those that reduce the binding of an antibody compound of the invention to CD47 by about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. Details of procedures for performing such competition assays are well known in the art and may be found, for example, in Harlow and Lane (1988) Antibodies, a Laboratory Manual [ Antibodies: a laboratory manual ], cold spring harbor laboratory press, cold spring harbor, new york. Such assays can be quantitative by using purified antibodies. A standard curve is established by titrating one antibody against itself, i.e. the same antibody is used for both the label and the competitor. The ability of unlabeled competitive monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labeled molecule to the plate was titrated. These results are plotted and the concentrations required to achieve the desired degree of binding inhibition are compared.

By these methods, in conjunction with the methods described in the examples below, it can be determined whether a mAb or antigen-binding fragment thereof that competes with an antibody compound of the present disclosure in such competition assays has the same or similar functional properties as an antibody compound of the present invention. In various embodiments, a competitive antibody for use in a therapeutic method encompassed herein has a biological activity as described herein in the range of about 50% to about 100% or about 125% or more as compared to an antibody compound disclosed herein. In some embodiments, the competing antibody has about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or the same biological activity as the antibody compound disclosed herein, as determined by the methods disclosed by the examples presented below.

The mAb or antigen-binding fragment thereof, or competitive antibody suitable for use in these compositions and methods, can be of any isotype described herein. Furthermore, any of these isoforms may comprise additional amino acid modifications as follows.

The monoclonal antibody or antigen-binding fragment thereof, or competitive antibody described herein, can be of the human IgG1 isotype.

The human IgG1 constant region of a monoclonal antibody, antigen-binding fragment thereof, or a competitive antibody described herein can be modified to alter antibody half-life. Antibody half-life is regulated in large part by Fc-dependent interactions with neonatal Fc receptors (ropenian and Alikesh, 2007). The human IgG1 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibody may be modified to increase half-life, including but not limited to the amino acid sequences N434A, T307A/E380A/N434A (Petkova et al, 2006, Yeung et al, 2009); M252Y/S254T/T256E (Dall' Acqua et al, 2006); T250Q/M428L (Hinton et al, 2006); and M428L/N434S (Zalevsky et al, 2010).

Instead of increasing half-life, there are some cases where it is desirable to decrease half-life, such as reducing the likelihood of adverse events associated with high antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) antibodies (Presta 2008). The human IgG1 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibody described herein may be modified to reduce half-life and/or reduce endogenous IgG, including but not limited to the amino acid modification I253A (Petkova et al, 2006); P257I/N434H, D376V/N434H (Datta-Mannan et al, 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al, 2005).

The human IgG1 constant region of a monoclonal antibody, antigen-binding fragment thereof, or a competitive antibody described herein, can be modified to increase or decrease antibody effector function. These antibody effector functions include, but are not limited to, Antibody Dependent Cellular Cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Phagocytosis (ADCP), C1q binding, and altered binding to Fc receptors.

The human IgG1 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibody described herein may be modified to increase antibody effector function, including but not limited to the amino acid modifications S298A/E333A/K334(Shields et al, 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al, 2006); F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al, 2007); G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al, 2008); K326A/E333A, K326A/E333S and K326W/E333S (Idusogene et al, 2001); S267E and S267E/L328F (Smith et al, 2012); H268F/S324T, S267E/H268F, S267E/S234T, and S267E/H268F/S324T (Moore et al, 2010); S298G/T299A (Sazinsky et al, 2008); E382V/M428I (Jung et al, 2010).

Monoclonal antibodies, antigen-binding fragments thereof, or the human IgG1 constant region of the competitive antibodies described herein may be modified to reduce antibody effector function, including but not limited to amino acid modifications N297A and N297Q (Bolt et al, 1993, Walker et al, 1989); L234A/L235A (Xu et al, 2000); K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaert et al, 2008); C226S/C229S/E233P/L234V/L235A (McEarchern et al, 2007); S267E/L328F (Chu et al, 2008).

The human IgG1 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibody described herein can be modified to reduce antibody effector function, including but not limited to the amino acid modifications V234A/G237A (Cole et al, 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S, H268Q/A330R/V309R/P331R, H268/A330R/V309/R/P331R, E233R/A330R, E233R/R, E233R/A330R, E233R/P271/271 72, E271/R, E271/36271/R, E271/R A271/R, G271/R/36271/R/36271/R, E233/G237/H268/P271/A330, P238/E233/P271/A330, P238/G237/H268/P271, P238/G237/P271/A330, P238/E233/H268/P271/A330, P238/E271/P271/A330, P238/E233/H238/H271/P271/A330, P238/E271/P237/A330, P271/P237/P268/P271/A330, P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S, P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R, P238D/E233D/G237D/H268/P271D/A330D, P238D/E233D/G237D/H268D/P271D/A36330 (An 2009, Mimoto,2013, et al).

The monoclonal antibody or antigen-binding fragment thereof, or competitive antibody described herein, can be of the human IgG2 isotype.

The human IgG2 constant region of a monoclonal antibody, antigen-binding fragment thereof, or a competitive antibody described herein, can be modified to increase or decrease antibody effector function. These antibody effector functions include, but are not limited to, Antibody Dependent Cellular Cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC), Antibody Dependent Cellular Phagocytosis (ADCP), and C1q binding, as well as altered binding to Fc receptors.

The human IgG2 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibodies described herein can be modified to increase antibody effector function, including but not limited to the amino acid modifications K326A/E333S (Idusogene et al, 2001).

The human IgG2 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibody described herein can be modified to reduce antibody effector function, including but not limited to the amino acid modifications V234A/G237A (Cole et al, 1999); V234A, G237A, P238S, H268A, E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S, P238S/H268A, V234A/G237A/P238S/H268S/V309S/A330S/P331S, H268/S/V309S/P331S, H268S/A330/S/36309/P331S, H268/A330/V309/S/36271 72, H268/V309/P309/S, H268/S/36271 72, E233/S/P271 72/S/36271 72/S, H268/S/36271, H268/S/36271, H271/S/36271, H/S/36271, H/S, G237/H268/P271/A330, E233/G237/H268/P271/A330, P238/E233/P271/A330, P238/G237/H268/P271, P238/G237/H268/P271, P238/G237/P271/A330, P238/G271/P271/A330, P238/E233/H268/P271/A330, P238/E233/P271/P233/P271/H268/A330, P238/E233/H271/H268/P268, P238/E271/H271/A330, P271/H271/P330, P271/, P238D/E233D/H268D/P271D/a 330D, P238D/G237D/H268D/P271D/a 330D, P238D/G237D/H268D/P271D/a 330D, P238D/E233D/G237D/H268/P271D/a D, P238D/E233D/G271 72/H271D/P271D/a D/P271D/P.

Monoclonal antibodies, antigen-binding fragments thereof, or the human IgG2 constant regions of competitive antibodies described herein may be modified to alter isotype and/or agonistic activity, including, but not limited to, amino acid modifications C127S (CH1 domain), C232S, C233S, C232S/C233S, C236S, and C239S (White et al, 2015, light et al, 2010).

The monoclonal antibody or antigen-binding fragment thereof, or competitive antibody described herein, can be of the human IgG3 isotype.

A human IgG3 constant region of a monoclonal antibody, or antigen binding fragment thereof, wherein said human IgG3 constant region of a monoclonal antibody, or antigen binding fragment thereof, can be modified at one or more amino acids to increase antibody half-life, Antibody Dependent Cellular Cytotoxicity (ADCC), Complement Dependent Cytotoxicity (CDC), or apoptotic activity.

A human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof, wherein the human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof, may be modified at amino acid R435H to increase antibody half-life.

The monoclonal antibody or antigen-binding fragment thereof, or competitive antibody described herein, can be of the human IgG4 isotype.

The human IgG4 constant region of a monoclonal antibody, antigen-binding fragment thereof, or a competitive antibody described herein can be modified to reduce antibody effector function. These antibody effector functions include, but are not limited to, Antibody Dependent Cellular Cytotoxicity (ADCC) and Antibody Dependent Cellular Phagocytosis (ADCP).

The human IgG4 constant region of a monoclonal antibody, antigen-binding fragment thereof, or competitive antibody described herein can be modified to prevent Fab arm exchange and/or reduce antibody effector function, including but not limited to the amino acid modifications F234A/L235A (alegure et al, 1994); S228P, L235E, and S228P/L235E (Reddy et al, 2000).

The present disclosure describes synergistic combinations that can provide improved effectiveness as measured by total tumor cell number, duration of recurrence, other clinical efficacy; and other indices of patient health. Alternatively, a synergistic combination of therapeutic effects is comparable to the effectiveness of monotherapy, while reducing adverse side effects, such as damage to non-target tissues, immune status, and other clinical indicators. The synergistic combinations of the present invention target agents that inhibit or block CD47 function; and agents that are chemotherapeutic or anti-cancer agents. The combination can provide one or more combinations of the agent (more specifically the anti-CD 47 antibody) and a chemotherapeutic agent, such as an anthracycline from the chemotherapeutic class, a platinum class, a taxol, a topoisomerase inhibitor, an antimetabolite, an antitumor antibiotic, a mitotic inhibitor, and an alkylating agent.

As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more agents may be administered simultaneously; in some embodiments, such agents may be administered sequentially; in some embodiments, such agents are administered in overlapping dosing regimens.

As used herein, the term "synergistic" or "synergistic effect" refers to the interaction of two or more therapeutic regimens (e.g., two or more therapeutic agents) to produce a combined effect that is greater than the sum of their individual effects.

As used herein, the term "additive" or "additive effect" refers to the interaction of two or more treatment regimens (e.g., two or more therapeutic agents) used in combination produces the same total effect as the sum of the individual effects.

The term "tumor" as used herein refers to all neoplastic cell growth and proliferation (whether malignant or benign) as well as all pre-cancerous and cancerous cells and tissues.

The terms "cancer," "cancerous," and "tumor" are used herein without mutual exclusion.

The terms "cancer" and "cancerous" refer to or describe the physiological condition typically characterized by abnormal cell growth/proliferation in mammals. Examples of cancer include, but are not limited to, carcinoma, lymphoma (i.e., hodgkin's lymphoma and non-hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, leukemia, and other lymphoproliferative disorders, as well as different types of head and neck cancer.

The term "susceptible cancer" (or "susceptable cancer") as used herein refers to a cancer whose cells express CD47 and which responds to treatment with an antibody or antigen-binding fragment thereof, or a competing antibody or antigen-binding fragment thereof, of the present disclosure.

As used herein, the terms "treating" or "treatment" mean slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but do not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treatment" or the like refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological state after the disease has begun to develop.

As used herein, the term "effective amount" refers to an amount or dose of an antibody compound of the present disclosure that provides the desired treatment or prevention after administration to a patient or organ in a single or multiple doses.

The precise effective amount for any particular subject will depend upon its size and health, the nature and extent of its condition, and the therapeutic agent or combination of therapeutic agents selected for administration. An effective amount for a given patient can be determined by routine experimentation and is within the judgment of a clinician. A therapeutically effective amount of an antibody compound of the invention may also include an amount ranging from about 0.1mg/kg to about 150mg/kg, from about 0.1mg/kg to about 100mg/kg, from about 0.1mg/kg to about 50mg/kg, or from about 0.05mg/kg to about 10mg/kg in each single dose administered to the harvested organ or patient. Antibody-based drugs are known to provide guidance in this regard. For example, Herceptin is administered by intravenous infusion of a 21mg/ml solution^TMWherein the initial loading dose is 4mg per kg body weight and the weekly maintenance dose is 2mg per kg body weight; rituxan^TMOnce weekly at 375mg/m 2; for example.

A therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compound on tumor regression, circulating tumor cells, tumor stem cells, or anti-tumor response. Analysis of the data obtained by these methods allows for modification of the treatment regimen during treatment such that optimal amounts of the antibody compounds of the disclosure are administered (whether alone or in combination with each other, or with another therapeutic agent, or both), and such that the duration of treatment can also be determined. In this manner, the dosing/treatment regimen can be modified over the course of therapy such that the minimum amount of antibody compound (used alone or in combination) that exhibits satisfactory results is administered and such that administration of such compound is continued for as long as is necessary to successfully treat the patient. Antibody-based drugs are known to provide guidance as to the frequency of administration, e.g., whether the drug should be administered daily, weekly, monthly, etc. The frequency and dosage may also depend on the severity of the symptoms.

In some embodiments, the antibody compounds of the present disclosure may be used as medicaments in human and veterinary medicine administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intraperitoneal, intracapsular, intraventricular, transdermal, topical, subcutaneous, intratumoral, intranasal, enteral, sublingual, intravaginal, intravascular or rectal routes. The composition may also be administered directly into the lesion (e.g., tumor). The dose treatment may be a single dose schedule or a multiple dose schedule. A hypodermic syringe may also be used to administer the pharmaceutical composition. Typically, these therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection may also be prepared. Veterinary applications include treatment of companion/pet animals such as cats and dogs; working animals such as pilot or service dogs, and horses; sports animals, such as horses and dogs; zoo animals, such as primates, felines (e.g., lions and tigers), felines, and the like; and other rare animals kept in captivity.

Such pharmaceutical compositions may be prepared by methods well known in the art. See, e.g., Remington, the science and Practice of Pharmacy [ hammton: pharmaceutical science and practice ], 21 st edition (2005), Lippincott Williams & Wilkins (lippocott Williams Wilkins publishing company), philadelphia, state of pennsylvania, and includes one or more antibody compounds disclosed herein, and a pharmaceutically or veterinarily acceptable carrier, diluent, or excipient, for example, physiologically acceptable.

The disclosure describes anti-CD 47 mAbs with different functional profiles, the antibodies having different combinations of properties selected from the group consisting of 1) exhibiting cross-reactivity with one or more CD47 species homologs, 2) blocking the interaction between CD47 and its ligand SIRP α, 3) increasing phagocytosis of human tumor cells, 4) inducing death of susceptible human tumor cells, 5) not inducing cell death of human tumor cells, 6) not having reduced or minimal binding to human red blood cells (hRBC), 7) having reduced binding to hRBC, 8) having minimal binding to hRBC, 9) causing reduced hRBC agglutination, 10) not causing detectable hRBC agglutination, 11) inhibition of the Nitric Oxide (NO) pathway 1, 12) not reversing TSP1 inhibition of the NO pathway, 13) causing loss of mitochondrial membrane potential, 14) not causing loss of mitochondrial potential, 15) causing increased release of human tumor cells on the surface of human tumor cells, 3526, 24, 16) causing increased release of human tumor cells, 16) and 3634, 16) increased endothelial cell expression of human endothelial cells, 16, 24, and 24, and 24, and 24, and 24, 5.

The anti-CD 47 antibodies and antigen-binding fragments thereof of the present disclosure have a combination of properties that differ from those of prior art anti-CD 47 antibodies. These features and characteristics will now be described in further detail.

As used herein, the term "binds human CD 47" refers to binding with an apparent Kd of greater than 50nM, for example, in a solid phase ELISA assay or a cell-based assay.

As used herein, the term "apparent binding affinity and apparent Kd" is determined by non-linear fitting (Prism GraphPad software) of binding data at different antibody concentrations.

Binding to CD47 of different species

The anti-CD 47 antibodies and antigen-binding fragments thereof of the present disclosure bind to human CD 47. In certain embodiments, the anti-CD 47 antibody exhibits cross-reactivity with one or more CD47 species homologs (e.g., CD47 homologs of non-human primate origin). In certain embodiments, anti-CD 47 antibodies and antigen-binding fragments thereof of the present disclosure bind to human CD47 and bind to CD47 of non-human primate, mouse, rat, and/or rabbit origin. Cross-reactivity with other species homologs can be particularly advantageous in the development and testing of therapeutic antibodies. For example, preclinical toxicity testing of therapeutic antibodies is frequently performed in non-human primate species, including but not limited to cynomolgus monkeys, green monkeys, rhesus monkeys, and squirrel monkeys. Cross-reactivity with homologues of these species may therefore be particularly advantageous for the development of antibodies as clinical candidates.

As used herein, the term "cross-reactive with one or more CD47 species homologs" refers to binding with an apparent Kd of greater than 50 nM.

Blocking the interaction between CD47 and SIRP α and promoting phagocytosis

CD47, also known as integrin-associated protein (IAP), is a 50kDa cell surface receptor consisting of: an extracellular N-terminal IgV domain, a five-transmembrane domain (five-transmembrane), and an alternatively spliced short C-terminal intracellular tail region.

Two ligands bind CD47, signal regulatory protein α (SIRP α) and platelet responsive protein-1 (TSP 1). TSP1 is present in plasma and is synthesized by a number of cells, including platelets SIRP α is expressed on hematopoietic cells including macrophages and dendritic cells.

Interaction of CD47 with SIRP α effectively sends a "do not eat me" signal to phagocytes (Oldenborg et al Science 288:2051-2054, 2000). in the case of therapy, blocking the interaction of SIRP α with CD47 by anti-CD 47mAb can provide an effective anti-cancer therapy by promoting uptake and elimination of cancer cells by the host immune system, thus, an important functional feature of some anti-CD 47 mAbs is the ability to block the interaction of CD47 with SIRP α such that macrophages phagocytose tumor cells expressing CD 47. several anti-CD 47 mAbs have been shown to block the interaction of CD47 with SIRP 9, including B6H12(Seiffert et al Blood 94:3633, 3643, 19826; Raberl et al Blood laid open No. 11J 25H 92, 3651, 11. mu.51, 11. mu. A. 11. A. Bu et al, 11. mu. A. Bu et al, 11. A. furthermore, A. Bu. A. 20. A. was shown by such as A. such as well as A. phagocytes, such as A. a Blood phagocytes, a Blood cells of phagocytes of CD11, a Blood cells of CD 35, a Blood cells of a Blood cells, a Blood laid down, a. a Blood cells of a Blood laid down. a Blood cells of a Blood laid down. a Blood cells of a Blood laid down, a Blood laid down [ 11, a Blood laid down [ 12, a. a Blood laid down [ 11, a Blood laid down [ 12A. a Blood laid down [ 11, a. a Blood laid down [ 11, a Blood laid down. a Blood laid down [ 11, a. a Blood laid down, a. a Blood laid down. a Blood laid down, a Blood laid.

As used herein, the term "blocking the binding of SIRP α to human CD 47" refers to a greater than 50% reduction in the binding of SIRP α -Fc to CD47 on cells by anti-CD 47mAb as compared to untreated cells or cells treated with negative antibodies.

The anti-CD 47 mabs described herein of the present disclosure block the interaction of CD47 with SIRP α and increase phagocytosis of human tumor cells.

"phagocytosis" of cancer cells refers to the engulfment and digestion of such cells by macrophages, and the eventual digestion or degradation of these cancer cells and the release of the digested or degraded cellular components in an extracellular or intracellular manner for further processing.

As used herein, the term "increase phagocytosis of human tumor cells" refers to a more than 2-fold increase in phagocytosis of human tumor cells by human macrophages in the presence of anti-CD 47mAb, as compared to untreated cells or cells treated with a negative control antibody.

Inducing death of tumor cells

Some soluble anti-CD 47 mabs initiated a cell death program upon binding to CD47 on tumor cells, causing a loss of mitochondrial membrane potential, a loss of ATP production capacity, an increase in cell surface expression of phosphatidylserine (detected by increased staining for annexin V), and cell death without caspase involvement or fragmentation of DNA. Such soluble anti-CD 47 mabs have potential for treatment of various solid and hematologic cancers. Some soluble anti-CD 47 mAbs have been shown to induce tumor cell death, including MABL-1, MABL-2, and fragments thereof (U.S. Pat. No. 8,101,719; Uno et al Oncorep. [ Oncology report]17:1189-94,2007; commun et al Biochem biophysis Res. [ Biochem and biophysical research communication ]]315:912-8,2004), Ad22(Pettersen et al J. Immunol. [ J. Immunol. ]]162:7031-7040, 1999; lamy et al J.biol.chem. [ J.Biol.Chem. ]]278:23915-]170:3544-3553, 2003; manna et al cancer research],64:1026-1036,2004). In previous analyses of MABL-1, MABL-2 and fragments thereof, Ad22 and 1F7, related methods were used to define apoptosis and cell death induced by these anti-CD 47 mabs. Annexin V and Propidium Iodide (PI) staining was assessed by flow cytometry to confirm that MABL scFV-15 dimer was early (annexin V)⁺，PI^-) And late (annexin V)⁺，PI⁺) Induction of apoptosis in CD 47-positive cells (Kikuchi et al biochem Biophys Res. Commun. [ Biochemical and biophysical research communication ]]315:912-8,2004). A similar approach was used to show that Ad22 induces apoptosis (annexin V)⁺，PI^-) And death (annexin V)⁺，PI⁺) Two-fold increase in cells (Pettersen et al j]162:7031-7040,1999). By analysis of annexin V by flow cytometry⁺Cell assessment 1F7 induction of apoptosis (Manna et al j]170:3544-3553,2003；Cancer Research by Manna et al],64:1026-1036,2004). Some of the anti-CD 47 mabs described herein of the present disclosure induce cell death of human tumor cells.

Phosphatidylserine exposure on the outer leaflet of the plasma membrane is widely observed during apoptosis and is the basis of annexin V binding assays to detect apoptotic cell death. It is important to note that in some systems, phosphatidylserine exposure and annexin V positive are reversible; i.e. some annexins V⁺Cells are viable and can regrow and reestablish phospholipid symmetry (Hammill et al exp. cell Res. [ experimental cell studies ]]251:16-21,1999). 7-Aminoactinomycin D (7-AAD) is a fluorescent intercalator that undergoes a spectral shift when associated with DNA. Live cells have intact membranes that exclude 7-AAD, whereas dead or apoptotic cells do not exclude 7-AAD.

The terms "induce cell death" or "killing" and the like are used interchangeably herein.

As used herein, the term "inducing death of human tumor cells" refers to increasing binding of annexin V (in the presence of calcium) and increasing uptake of 7-amino actinomycin D (7-AAD) or propidium iodide in response to treatment with anti-CD 47 mAb. These characteristics can be quantified by flow cytometry in three cell populations: annexin V positive (annexin V)⁺) Annexin V positive/7-AAD negative (annexin V)⁺/7-AAD^-) And annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺). Induction of cell death was defined as an increase of more than 2-fold for each of the above cell populations in human tumor cells caused by soluble anti-CD 47mAb compared to background obtained with negative control antibody, (humanized, isotype matched antibody) or untreated cells.

Another indicator of cell death is the loss of mitochondrial function and membrane potential of tumour cells, as determined by one of several available measurements (potentiometric fluorescent dyes, such as DiO-C6 or JC1, or formazan-based assays, such as MTT or WST-1).

As used herein, the term "causing a loss of mitochondrial membrane potential" refers to a statistically significant (p <0.05 or greater) reduction in mitochondrial membrane potential by soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or no treatment.

Inducing cell death refers to the ability of certain soluble anti-CD 47 antibodies, murine antibodies, chimeric antibodies, humanized antibodies, or antigen-binding fragments thereof (as well as competitive antibodies and antigen-binding fragments thereof) disclosed herein to kill cancer cells via a cell autonomous mechanism without the involvement of complement or other cells, including but not limited to T cells, neutrophils, natural killer cells, macrophages, or dendritic cells.

In the humanized or chimeric mabs of the invention, those mabs that induce cell death of human tumor cells cause an increase in annexin V binding similar to the findings reported for: anti-CD 47mAb Ad22(Pettersen et al J.Immunol. [ J.Immunol ]166: 4931-; 1F7(Manna and Frazier J. Immunol. [ J. Immunol ]170: 3544-; and MABL-1 and 2 (U.S. Pat. No. 7,531,643B 2; U.S. Pat. No. 7,696,325B 2; U.S. Pat. No. 8,101719B 2).

Cell viability assays are described in the NCI/NIH instruction manual, which describes various types of cell-based assays that can be used to assess the induction of cell death by CD47 antibody: "Cell Viability Assays", Dr. Terry L Riss, Richard A Moravec. Proc. Sci, Andrew L Niles. Lew., HeleneA Benik. Lew., Tracy J Worzella. Lew., and Lisa Minor Ph. Przeman information, published 5/1.2013.

Binding to hRBC

CD47 expressed on human erythrocytes (hRBC) (Brown.J. Cell Biol. [ J. Biobiol. J. 111:2785-2794, 1990; Avent.biochem J. [ J. bioch. J. ] (1988)251: 499-505; Knapp.blood ] (1989) vol. 74, No. 4, 1448-1450; Oliveira et Biophycaca [ biochemistry et Biophycaca ]1818: 481-490, 2012; Petrova P. et al. Cancer Res. 2015 [ Cancer research ] 2015; 75(15 Supplement) 4271. RBC-CD 47 bound to RBC-CD 47 including B6H12(Brown et J. Biol. Biol. Biot. Biotic. J. 2015. 19810; European Pat. No. 2015. No.: 2015. No. 2015. (1987; European Pat. No. 2015J. 2015. No. 2015.: 2015 et 2015.: 2015. 2015 et 2015J. 2015.: 2015. 2015J. 2015 et 2015. No. 2015 [ 12; No. 2015.: 2015J. No. 2015 et 35; No. 35; European No. 12; European No. 12; European No. 12. No. 35; European No. 35; European No. 2015 et seq.: No. 12; European No. 35; European No. 12; European No. 35; European No. 12; European No. 35; European No. 12; European No. 12; European No. 12; European No. 7; European No. 12; European No. 11; European No. 12; European No. 7; European No. 12; European No. 11; European No. 12; European No. 7; European No. 11; European No. 12; European No. 11; European No. 7; European No. 11; European No. 7; European No. 75; European No. 11; European No. 12; European No. 75; European No. 250; European No. 11.

As used herein, the terms "red blood cell(s)" and "red blood cell(s)" are synonymous and are used interchangeably herein.

As used herein, the term "reduced binding to hrbcs" means that the apparent Kd of the anti-CD 47mAb binding to hrbcs is 10-fold or greater than the apparent Kd on human tumor cells, wherein the tumor cells are OV10hCD47 cells (human OV10 ovarian cancer cell line expressing human CD 47).

As used herein, the term "unbound" or "NB" refers to binding to hrbcs that is not measurable at anti-CD 47mAb concentrations up to and including 50 μ g/ml.

Prior to the disclosure described herein, no anti-CD 47mAb was reported that did not bind to human RBCs expressing CD 47.

Some of the anti-CD 47 mabs disclosed herein have reduced or undetectable binding to human RBCs.

Binding to human endothelial cells and other normal human cells

In addition to being expressed/overexpressed on most hematologic malignancies and solid tumors (Willingham et al, proc. natl. acad. Sci. [ journal of the national academy of sciences ]2012), CD47 is also expressed by many, but not all, normal Cell types, including, but not limited to, RBCs (see previous sections), lymphocytes and monocytes, endothelial cells, and brain, liver, muscle cells and/or tissues (Brown et al, j.biol. [ journal of Cell biology ] 1990; Reinhold et al, j.cell Sci. [ journal of Cell science ] 1995; Matozaki et al, Cell [ Cell ] 2009; Stefanidakis et al, Blood [ Blood ] 2008; Xiao et al, Cancer Letters [ Cancer communication ] 2015). Due to this expression, it is expected that some anti-CD 47 mabs will bind to these normal cell types/tissues in addition to the cancer cells that are the target of therapy. Therefore, it is desirable to identify anti-CD 47 mabs that do not bind to or have reduced binding to some of these normal cells to reduce potential undesirable effects on these normal cells and also to allow more available antibodies to bind to tumor cells.

As used herein, the term "reduced and normal human cells (including but not limited to endothelial cells, epithelial cells, skeletal muscle cells, peripheral blood mononuclear cells, or CD3⁺T cells) "refers to the anti-CD 47mAb binding to these cellsThe apparent Kd of binding was 10-fold or more greater than the apparent Kd of the anti-CD 47mAb binding to a human tumor cell, wherein the tumor cell is OV10hCD 47.

As used herein, the term "unbound" or "NB" refers to non-measurable interactions with normal human cells (including but not limited to endothelial cells, epithelial cells, skeletal muscle cells, peripheral blood mononuclear cells, or CD 3) at concentrations of anti-CD 47mAb up to and including 30 μ g/ml⁺T cells).

Agglutination of RBC

Red Blood Cell (RBC) agglutination or hemagglutination is a homotypic interaction that occurs when RBCs agglutinate or clump when incubated with different reagents, including antibodies to RBC antigens and cell surface proteins such as CD 47. A number of anti-CD 47 antibodies have been reported to cause hemagglutination of isolated human RBCs in vitro in a concentration-dependent manner, including B6H12, BRIC126, MABL-1, MABL-2, CC2C6, and 5F9(Uger R. et al Cancer Res. [ Cancer research ] 2014; 74(19 supplement): abstract nr 5011; U.S. Pat. No. 9,045,541; Uno et al Oncol Rep. [ oncology report ]17: 1189-. This functional effect requires binding of RBC by intact bivalent antibodies and can be reduced or eliminated by generating antibody fragments F (ab') or svFv (Uno et al Oncol Rep. [ Oncol report ]17: 1189-. Other functional properties of these fragments, including cell killing, have been shown to be reduced or retained in these fragments (Uno et al Oncorep [ Oncolor report ]17: 1189-. Mouse antibody 2D3 is an example of an anti-CD 47 antibody that binds to CD47 on red blood cells but does not cause hemagglutination (U.S. patent No. 9,045,541, Petrova et al Cancer Res. [ Cancer research ] 2015; 75(15 supplementary edition): abstract nr 4271).

Blood agglutination has been shown to reduce/eliminate selective binding to human RBCs but not other cells by using SIRP α -Fc fusion proteins (Uger r. et al Blood 2013; 122(21): 3935.) additionally, mouse anti-CD 47mAb 2a 1and the humanized version of 2a1 were reported to block CD47/SIRP α but did not exhibit hemagglutination activity (U.S. patent 9,045,541) a small group of mouse anti-human CD47 antibodies (3 in 23) were reported not to cause hemagglutination of human RBCs (Pietsch E et al Cancer Res. [ Cancer research ] 2015; 75(15 plemented version): abstract nr 2470.) thus, prior to the disclosure described herein, the identification of SIRP α D47 that blocks binding, does not have or reduces binding to and/or does not cause hemagglutination of human RBCs the term "RBC (hemagglutination)" refers to a type of hemagglutination, thus the term "hemagglutination" of cell type.

As used herein, the term "reduced hemagglutination" refers to measurable agglutinative activity of hrbcs at anti-CD 47mAb concentrations greater than 1.85 μ g/ml in a washed RBC assay, as well as an agglutinative activity that is not measurable at concentrations less than or equal to 1.85 μ g/ml.

As used herein, the term "undetectable hemagglutination" refers to the unmeasurable agglutination activity of hrbcs in a washed RBC assay at anti-CD 47mAb concentrations of greater than or equal to 0.3pg/mL to concentrations of less than or equal to 50 μ g/mL.

Some of the anti-CD 47 antibodies described herein cause reduced or undetectable hemagglutination of human RBCs.

Immunogenic cell death

The concept of Immunogenic Cell Death (ICD) has emerged in recent years. Unlike non-immunogenic cell death, this form of cell death stimulates an immune response against antigens from cancer cells. ICDs are induced by specific chemotherapeutic drugs, including anthracyclines (doxorubicin, daunorubicin, and mitoxantrone) and oxaliplatin, but not by cisplatin and other chemotherapeutic drugs. ICDs are also induced by bortezomib, cardiac glycosides, photodynamic therapy and radiation (Galluzi et al nat. rev. immunol. [ natural immunological review ]17:97-111,2016). ICDs of tumor cells are characterized by the release from or exposure to the surface of tumor cells of specific ligands: 1) pre-apoptotic cell surface exposure to calreticulin, 2) Adenosine Triphosphate (ATP) secretion, 3) release of high mobility group protein 1(HMGB1), 4) annexin a1 release, 5) type I interferon release and 6) C-X-C motif chemokine ligand 10(CXCL 10). These ligands are endogenous damage-associated molecular patterns (DAMPs) that include cell death-related molecules (CDAMs) (Kroemer et al annu. rev. immunol. [ annual review of immunology ]31:51-72,2013). Importantly, each of these ligands induced during ICD binds to a specific receptor, called Pattern Recognition Receptor (PRR), which contributes to the anti-tumor immune response. ATP binds to the purinergic receptor PY2, G-protein coupled 2(P2RY2) and PX2, ligand-gated ion channel 7(P2RX7) on dendritic cells, causing recruitment and activation of dendritic cells, respectively. Annexin a1 binds to formyl peptide receptor 1(FPR1) on dendritic cells, causing the dendritic cells to home. Calreticulin expressed on the surface of tumor cells binds to LRP1(CD91) on dendritic cells, thereby facilitating antigen uptake by dendritic cells. HMGB1 binds to toll-like receptor 4(TLR4) on dendritic cells, causing dendritic cell maturation. As a component of ICDs, tumor cells release type I interferons, resulting in signaling through the type I interferon receptor, and release CXCL10 that favors effector CXCR3+ T cell recruitment, the action of these ligands on their receptors facilitating DC recruitment into the tumor, phagocytosis of tumor antigens by the DC, and presentation of optimal antigens to T cells. Kroemer et al have suggested that the precise combination of the above mentioned CDAMs elicited by ICDs could overcome the mechanisms that normally prevent the activation of anti-tumor immune responses (Kroemer et al Annu Rev Immunol [ Annu. Rev. Immunol ]31:51-72,2013; Galluzi et al Nat. Rev. Immunol. [ Nature. Immunol ]17:97-111,2016). When mouse tumor cells treated in vitro with an ICD-induced form are administered in vivo to syngeneic mice, they provide effective vaccination resulting in an anti-tumor adaptive immune response, including memory. This vaccination effect could not be tested in xenograft tumor models because the mice used in these studies lack an intact immune system. The data available suggest that ICD effects induced by chemotherapy or radiation will promote adaptive anti-tumor immune responses in cancer patients. The components of ICDs are described in more detail below.

In 2005, it was reported that tumor cells that died in response to anthracycline chemotherapy in vitro elicited a potent anti-tumor immune response when administered in vivo in the absence of adjuvant (Casares et al j.exp.med. [ journal of experimental medicine ]202:16911701,2005). This immune response protects mice from subsequent re-challenge by viable cells of the same tumor and causes regression of established tumors. Anthracyclines (doxorubicin, daunorubicin, and idarubicin) and mitomycin C induce apoptosis of tumor cells with caspase activation, but apoptosis induced by anthracyclines alone leads to immunogenic cell death. Caspase inhibition does not inhibit cell death induced by doxorubicin, but inhibits the immunogenicity of tumor cells that die in response to doxorubicin. The major role of dendritic cells and CD8+ T cells in the immune response elicited by apoptotic tumor cells treated with doxorubicin was established by the following demonstration: depletion of these cells abrogates the immune response in vivo.

Calreticulin is one of the most abundant proteins in the Endoplasmic Reticulum (ER) calreticulin has been shown to be rapidly translocated from the ER lumen to the surface of cancer cells in a pre-apoptotic form in response to various ICD inducers, including anthracyclines (Obeid et al Nat Med [ nature ]13:54-61,2007; Kroemer et al annu.rev.immumol. [ annual review of immunology ]31:51-72,2013.) blockade and knockdown of calreticulin inhibits dendritic Cell phagocytosis of anthracycline-treated tumor cells and eliminates their immunogenicity in mice the exposure of calreticulin by anthracyclines or oxaliplatin is activated by an ER stress response that includes phosphorylation of the eukaryotic translation initiation factor eIF2 α by PKR-like ER kinases including "eat me" signaling (garda et al [ Cell ]123: 2005) and without phagocytosis of dendritic Cell initiation factor kinase antigen on eukaryotic cells (CD91) and thus results in overall in a response to CD 62 antigen presentation by macrophage expressed by macrophage cells expressing the inflammatory response to CD2 expressed by macrophage phagocytosis stimulating cytokine targeting CD 2.

In addition to calreticulin, the protein disulphide isomerase A3(PDIA3) (also known as Erp57) has been shown to translocate from the ER to the surface of tumor cells following treatment with mitoxantrone, oxaliplatin and irradiation with UVC light (Panaretakis et al Cell Death Differ [ Cell Death and differentiation ]15: 1499-. Human ovarian Cancer cell lines, primary ovarian Cancer cells, and human prostate Cancer cell lines express cell surface calreticulin, HSP70, and HSP90(Fucikova et al Cancer Res [ Cancer research ]71:4821-4833,2011) following treatment with anthracycline doxorubicin and idarubicin. HSP70 and HSP90 bind PRR LRP1 on antigen presenting cells; the PRR to which PDIA3 binds has not been identified (Galluzi et al nat. Rev. Immunol. [ annual review of immunology ]17:97-111,2016).

TLR4 has been shown to be required for cross-presentation of dead tumor cells and to control the processing and presentation of tumor antigens. Among the proteins known to bind and stimulate TLR4, HMGB1 is uniquely released by mouse tumor cells, with ICD induced by irradiation or doxorubicin (Apetoh et al nat. med. [ nature medicine ]13: 1050-. The efficient induction of anti-tumor immunity in vivo by doxorubicin therapy of mouse tumor cells requires the presence of HMGB 1and TLR4, as demonstrated by the abrogation of the immune response by inhibiting HMGB 1and knocking out TLR 4. These preclinical findings are clinically relevant. Breast cancer patients carrying the TLR4 loss of function allele relapse more rapidly following radiation therapy and chemotherapy than those carrying the normal TLR4 allele.

Ghiringhelli et al demonstrated that mouse tumor cells treated in vitro with oxaliplatin, doxorubicin and mitoxantrone release ATP and that ATP binds to the purinergic receptor PY2, G-protein coupled 2(P2RY2) and PX2 on dendritic cells, ligand-gated ion channel 7(P2RX7) (Ghiringhelli et al Nat Med [ Nature medicine ]15:1170-1178, 2009.) binding of ATP to P2RX7 on DCs triggers the NOD-like receptor family, the pyrin domain-dependent caspase-1 activation complex (inflammasome) containing 3 proteins (NLRP3) allowing secretion of interleukin-1 β (IL-1 RY β) which is essential for initiating interferon γ -producing CD8+ T cells by dying tumor cells, thus ATP-induced IL-1 β appears to be immune system-aware of one of the cell death-induced chemotherapeutics as an immunogen-derived tumor cell agonist and further has a negative effect on tumor cell survival rate in the tumor-mediated clinical trial of tumor growth of tumor cells [ HLA-2: HLA-96: 9-II ] and the presence of a prognostic factor for tumor cells in the tumor-mediated clinical tumor cells (HLA-7: NO: 11: NO: 7: NO:7, 2, and NO: 7: NO

Michaud et al demonstrated that autophagy is required for immunogenicity of chemotherapy-induced cell death (Michaud et al Science 334:1573-1577, 2011). Release of ATP from dead tumor cells requires autophagy and mouse tumors with autophagic activity, rather than autophagy-deficient mouse tumors, attract dendritic cells and T lymphocytes into the tumor microenvironment in response to ICD-inducing chemotherapy.

Ma et al solved the problem of how chemotherapy-induced cell death leads to efficient antigen presentation to T cells (Ma et al Immunity [ immunology ]38:729-741, 2013). They found that a specific class of tumor infiltrating lymphocytes, CD11c + CD11b + Ly6Chi cells, is particularly important for inducing anti-cancer immune responses by anthracyclines. ATP released by the dead cancer cells recruits bone marrow cells into the tumor and stimulates local differentiation of CD11c + CD11b + Ly6Chi cells. These cells have been shown to be particularly effective in capturing and presenting tumor cell antigens and, after adoptive transfer to naive mice, confer protection against challenge with live tumor cells of the same cell line.

Anthracyclines have been shown to stimulate tumor cells to rapidly produce type I interferons upon TLR3 activation (situgu et al nat. med. [ natural medicine ]20: 1301-.

Another receptor on dendritic cells involved in chemotherapy-induced anti-cancer immune responses has recently been identified: formyl peptide receptor-1, which binds to annexin A1(Vacchelli et al Science 350:972-978, 2015). One screen was devised by vaccheli et al to identify candidate genetic defects that negatively affected the response to chemotherapy. They identified loss of function alleles of the gene encoding formyl peptide receptor 1(FPR1) that correlate with poor metastasis-free survival and overall survival in breast and colorectal cancer patients receiving adjuvant chemotherapy. The therapeutic effect of anthracyclines is abolished in tumor-bearing Fpr 1-/-mice due to impaired anti-tumor immunity. FPR 1-deficient DCs do not approach dead tumor cells and, therefore, do not elicit anti-tumor T cell immunity. Two anthracyclines, doxorubicin and mitoxantrone, stimulate the secretion of annexin a1 (one of the four known ligands of FPR 1). FPR 1and annexin a1 promote stable interactions between dead cancer cells and human or mouse leukocytes.

In addition to anthracyclines and oxaliplatin, other drugs have been shown to induce immunogenic cell death. Cardiac glycosides, including clinically used digoxin and digitoxin, have also been shown to be potent inducers of immunogenic cell death of tumor cells (Menger et al Sci Transl Med [ scientific transformation medicine ]4:143ra99,2012). Other chemotherapeutic agents and cancer drugs reported to induce the expression or release of DAMP are bleomycin, bortezomib, cyclophosphamide, paclitaxel, vorinostat (vorinostat) and cisplatin (Garg et al front. immunol. [ immunological frontier ]588:1-24,2015; Menger et al sci. trans. med. [ scientific transformation medicine ]4:143ra99,2012; Martins et al Oncogene [ Oncogene ]30: 1147-. Importantly, these results are clinically relevant. Administration of digoxin during chemotherapy has a significant positive impact on overall survival in patients with breast, colorectal, head and neck, and hepatocellular carcinoma, but fails to improve overall survival in patients with lung and prostate cancer.

The anti-CD 20 monoclonal antibody rituximab has improved outcomes in a variety of B cell malignancies. The success of rituximab (also known as the type I anti-CD 20 mAb) led to the development of type II anti-CD 20 mabs (including atuzumab and tositumomab). Cheadle et al studied the induction of immunogenic cell death by anti-CD 20mAb (Cheadle et al Brit.J.Haematol. [ J.Henkel. Hematology. 162:842-862, 2013). They found that the cell death induced by adalimumab and tositumomab is a form of immunogenic cell death characterized by the release of HMGB1, HSP90 and ATP. Type I anti-CD 20mAb did not cause release of HMGB1, HSP90 and ATP. Incubation of the supernatant from the human tumor cell line treated with trastuzumab resulted in maturation of human dendritic cells, consistent with the previously described effects of HMGB 1and ATP on dendritic cells. In contrast to the results reported by Cheadle et al, Zhao et al reported that both type I and type II anti-CD 20 mAbs increased HMGB1 release from human diffuse large B-cell lymphoma cell lines, but did not cause ATP release or cell surface expression of calreticulin (Zhao et al Oncotarget [ tumor target ]6:27817-27831, 2015).

DAMP calreticulin, ATP, HMGB1, annexin a1, type I interferon release, CXCL10, PDIA3, HSP70 and/or HSP90 have not been reported to be released from or exposed on the surface of tumor cells in response to anti-CD 47 mAb. As disclosed herein, the anti-CD 47mAb caused the release or exposure of the above listed DAMPs from the tumor cell surface (characteristic of ICDs), an unexpected result. These DAMPs are expected to promote therapeutically beneficial adaptive anti-tumor immune responses. Combining the anti-CD 47mAb that causes the release/expression of DAMP with a chemotherapeutic agent that causes an immunogenic cell death effect can result in greater therapeutic benefit than using the agent alone.

As disclosed herein, "causing an increase in cell surface calreticulin expression of human tumor cells" refers to a statistically significant increase in calreticulin expression (p <0.05 or greater) by the soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or no treatment.

As disclosed herein, the term "release of … …" is synonymous with secretion and is defined as the extracellular appearance of ATP, HMGB1, annexin a1, type I interferon and CXCL 10.

As disclosed herein, "causing an increase in adenosine triphosphate release from human tumor cells" refers to a statistically significant increase in ATP (p <0.05 or greater) in the supernatant by the soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or no treatment.

As disclosed herein, "causing an increase in high mobility group family protein 1 release of human tumor cells" refers to a statistically significant increase (p <0.05 or greater) of HMGB1 in the supernatant compared to background obtained with negative control, humanized isotype-matched antibody, or untreated soluble anti-CD 47 mAb.

As disclosed herein, "causing an increase in type I interferon release from human tumor cells" refers to a statistically significant increase (p <0.05 or greater) in type I interferon or type I interferon mRNA in the supernatant by the soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or no treatment.

As disclosed herein, "causing an increase in C-X-C motif chemokine ligand 10(CXCL10) release from human tumor cells" refers to a statistically significant increase (p <0.05 or greater) in CXCL10 or CXCL10 mRNA in the supernatant by soluble anti-CD 47mAb compared to background obtained with negative control, humanized isotype-matched antibody, or untreated.

As disclosed herein, "causing an increase in cell surface PDIA3 expression of human tumor cells" refers to a statistically significant increase in PDIA3 expression (p <0.05 or greater) by soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or untreated.

As disclosed herein, "causing an increase in cell surface HSP70 expression of human tumor cells" refers to a statistically significant increase in HSP70 expression (p <0.05 or greater) by the soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or no treatment.

As disclosed herein, "causing an increase in cell surface HSP90 expression of human tumor cells" refers to a statistically significant increase in HSP90 expression (p <0.05 or greater) by the soluble anti-CD 47mAb compared to background obtained with negative controls, humanized isotype-matched antibodies, or no treatment.

pH dependence of anti-CD 47mAb binding

Most antibody binding (particularly in the blood compartment and against normal cells) occurs at physiological pH (pH 7.2-7.4). Therefore, the binding affinity of therapeutic mabs is usually assessed in vitro at physiological pH. However, the Tumor Microenvironment (TME) is more acidic in nature with a pH below 7.0. This appears to be due to a variety of differences, including hypoxic, anaerobic glycolysis leading to lactate production and ATP hydrolysis (Tannock and Rotin, Cancer Res [ Cancer research ] 1989; Song et al, Cancer Drug Discovery and Development [ Cancer Drug Discovery and Development ] 2006; Chen and Pagel, Advan Radiol [ advanced radiology ] 2015). Acidic pH can provide an advantage to tumors by promoting invasiveness, metastatic manifestation, chronic autophagy, resistance to chemotherapy, and reduced efficacy of immune cells in the tumor microenvironment (Estrella et al, Cancer Res [ Cancer studies ] 2013; Wojtkowiak et al, Cancer Res [ Cancer studies ], 2012; Song et al, Cancer Drug Discovery and Development [ Cancer Drug Discovery and Development ], 2006; Barar, journal of BioImpacts, 2012). However, identification of anti-CD 47 antibodies with the property of increased binding affinity at acidic pH would confer a therapeutic advantage of higher binding to CD47 on tumor cells within acidic TME compared to normal cells. Antibodies with pH-dependent properties have been generated with the aim of recovering the antibody. However, in contrast to the property of exhibiting enhanced binding at acidic pH, these antibodies bind their target antigen with high binding capacity at physiological pH, but release their target at acidic pH (Bonvin et al, mAbs 2015; Igawa and Hattori, Biochem Biophys Acta [ Biochem Biophys Acta ] 2014).

As disclosed herein, "has greater affinity for CD47 at acidic pH than at physiological pH" means that the apparent Kd at acidic pH (<7.2) is increased by 5-fold or more compared to physiological pH (7.2-7.4).

Combination of functional characteristics

In some embodiments of the anti-CD 47 antibodies described herein, the combination of properties not exhibited by prior art anti-CD 47 antibodies suggested for human therapeutic use is also characterized. Accordingly, the anti-CD 47 antibodies described herein are characterized by:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells; and

d. inducing death of susceptible human tumor cells.

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of susceptible human tumor cells; and

e. no detectable human red blood cell (hRBC) agglutination is caused.

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of susceptible human tumor cells; and

e. causing a reduction in human red blood cell (hRBC) agglutination.

a. specifically binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of susceptible human tumor cells; and

e. with reduced hRBC binding.

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. does not cause detectable human red blood cell (hRBC) agglutination; and

e. with minimal binding to hrbcs.

a. specifically binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. does not cause detectable human red blood cell (hRBC) agglutination; and

e. with reduced hRBC binding.

In the examples described herein, an anti-CD 47 monoclonal antibody or antigen binding fragment thereof may additionally have one or more of 1) exhibit cross-reactivity with one or more species homologs of CD47, 2) block the interaction between CD47 and its ligand SIRP α, 3) increase phagocytosis of human tumor cells, 4) induce apoptosis of susceptible human tumor cells, 5) do not induce cell death of human tumor cells, 6) do not have reduced or minimal binding to human red blood cells (hRBC), 7) have reduced binding to hRBC, 8) have minimal binding to hRBC, 9) cause reduced hRBC agglutination, 10) do not cause detectable hRBC agglutination, 11) reverse TSP1 inhibition of the Nitric Oxide (NO) pathway, 12) do not reverse TSP1 inhibition of the NO pathway, 13) cause loss of mitochondrial membrane potential, 14) do not cause loss of mitochondrial membrane potential, 15) cause loss of mitochondrial membrane potential on human tumor cells, 15) cause increased release of calcium triphosphate on human tumor cells, 47, or peripheral vascular endothelial cells, 16) do not cause increased release of human endothelial cells, 16) and human endothelial cells, 16) cause increased expression of human endothelial cells, 2) of human endothelial cells, 16, 24, and 3620, 24, A3, a high, a high, a high, a high, a human tumor cell expression of human endothelial cell expression of.

In some embodiments, a monoclonal antibody or antigen binding fragment thereof is provided that binds to human CD47, blocks binding of SIRP α to human CD47, increases phagocytosis of human tumor cells, and induces death of human tumor cells, wherein the monoclonal antibody or antigen binding fragment thereof exhibits pH-dependent binding to CD47 present on the cell, in other embodiments, the disclosure provides a monoclonal antibody or antigen binding fragment thereof that binds to human CD47, blocks binding of SIRP α to human CD47, increases phagocytosis to human tumor cells, and induces death of human tumor cells, wherein the monoclonal antibody or antigen binding fragment thereof exhibits reduced binding to normal cells.

CD47 antibody

Many human cancers up-regulate Cell surface expression of CD47, and those expressing the highest levels of CD47 appear to be the most aggressive and most lethal to patients, increased CD47 expression is thought to protect cancer cells from phagocytosis by sending a "do not eat me" signal to macrophages via SIRP α, an inhibitory receptor that prevents phagocytosis of CD 47-bearing cells (Oldenborg et al Science 288:2051-2054, 2000; Jaiswal et al 2009) Cell [ 138(2):271-85 l; Chao et al (2010) Science transformational Medicine [ 63 ]: 63ra 94.) thus, increased CD47 expression for many cancers provides them with "self-masking" slowing their clearance by macrophages and dendritic cells.

Such blocking anti-CD 47 mAbs exhibiting this property increase macrophage phagocytosis of Cancer cells, which may reduce tumor burden (Majeti et al (2009) Cell [ Cell ]138(2): 286-99; US 9,045,541; Willingham et al (2012) Proc Natl Acad.Sci.USA [ Proc. Natl. Acad. Sci. USA ]109(17): 6662-.

anti-CD 47 mAbs have also been shown to promote adaptive immune responses to tumors in vivo (Tseng et al (2013) PNAS 110(27): 11103-.

However, the mechanism by which anti-CD 47mAb can attack transformed cells in Cancer therapy has not been elucidated, several groups have shown that specific anti-human CD47mAb induces cell death of human tumor cells [ Pettersen et al J.Immunol ]166: 4931. 4942, 2001; Lamy et al J.biol.Chem. [ biochem ]278: 23915. 2003 ] AD22 has shown to induce rapid mitochondrial dysfunction and rapid cell death with early phosphatidylserine exposure and reduced mitochondrial membrane potential (Lamy et al J.biol.Chem. [ biochem ]278: 23915. 23921,2003) that anti-CD 47 MABL-2 and its fragments can induce cell death of human leukemia cell lines but not normal cells in vitro and have a selective anti-human tumor cell killing effect against CD-5926, CD-3527, which is directly linked to the effector receptor effector (mAb) and which is directly linked to the effector receptor effector (mAb) receptor effector receptor.

An additional mechanism by which anti-CD 47 mabs may be used in cancer therapy is by promoting anti-tumor immune responses. The discovery that anti-CD 47mAb causes tumor cells to release DAMPs that cause DC maturation, activation and homing, and T cell attraction, links anti-CD 47mAb treatment to the development of a therapeutically desirable anti-tumor immune response. The anti-CD 47mAb was not previously shown to cause tumor cell release of ATP, HMGB1, annexin a1, type I interferon and CXCL10 and tumor cell expression of calreticulin, PDIA3, HSP70 and HSP 90.

After the ischemic phase of the tissue, the onset of blood flow causes a damage known as "ischemia reperfusion injury" or IRI. IRI is a cause of poor outcome in many surgical procedures, where IRI occurs due to the inevitable cessation of blood flow for a period of time in many forms/causes of trauma (where blood flow is interrupted and later restored by therapeutic intervention) and in procedures requiring organ transplantation, heart/lung bypass surgery, reattachment of severed body parts, plastic and cosmetic surgery, and other situations involving cessation and resumption of blood flow. Ischemia itself causes many physiological changes, by which it will ultimately lead to cell and tissue necrosis and death. Reperfusion causes its own set of damaging events including reactive oxygen species generation, thrombosis, inflammation and cytokine mediated damage. These pathways, limited by the TSP1-CD47 system, are precisely those that would be most beneficial in combating IRI damage, including the NO pathway. Thus, blocking the TSP1-CD47 pathway as with the antibodies disclosed herein would provide more robust function for the endogeneous protection pathways. anti-CD 47mAb has been shown to reduce organ damage in animal models of renal warm ischemia (renal ischemia) (Rogers et al J Am Soc Nephrol. [ J. Am. renopathy Act. 23: 1538. cndot. 1550,2012), hepatic ischemia reperfusion injury (Isenberg et al Surgery. 144: 752. 761,2008), renal Transplantation (Lin et al Transplantation. 98: 394. 2014; Rogers et al Kidney International. International [ International ]90: 334. cndot. 347,2016) and Liver Transplantation (including fatty Liver degeneration) (Xiao et al Liver Transpl. Liver Transplantation [ 21: 468. cndot. 477,2015; Xiao et al Transplantation [ 100: 1480. cndot. 1489,2016). In addition, anti-CD 47mAb caused a significant reduction in right ventricular systolic pressure and right ventricular hypertrophy in the monocrotaline model of pulmonary hypertension (Bauer et al Cardiovasc Res [ cardiovascular studies ]93: 682-. Studies in the flap model showed that modulation of CD47 (including the use of anti-CD 47 mAb) inhibited TSP 1-mediated CD47 signaling. This leads to an increase in the activity of the NO pathway, which leads to a decrease in IRI (Maxhimer et al plant Reconster Surg [ plastic and reconstructive surgery ]124:1880-

anti-CD 47mAb has also been shown to be effective in other models of cardiovascular disease. In a mouse aortic constriction model of pressure-loaded left ventricular Heart failure, the anti-CD 47mAb reduced cardiomyocyte hypertrophy, reduced left ventricular fibrosis, prevented left ventricular weight gain, reduced ventricular stiffness, and normalized the change in pressure-volume closed curve (Sharifi-Sanjani et al J Am Heart assist [ journal of the american Heart association ], 2014). anti-CD 47mAb improves atherosclerosis in various mouse models (Kojima et al Nature, 2016).

Indications for cancer

Presently disclosed are anti-CD 47 mabs and antigen-binding fragments thereof that are effective as cancer therapeutics, preferably administered parenterally to patients with susceptible hematologic and solid cancers, including but not limited to leukemia, including systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T-cell-ALL, Acute Myelogenous Leukemia (AML), myelogenous leukemia, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), myeloproliferative disorders/neoplasms, monocytic leukemia, and plasma cell leukemia; multiple Myeloma (MM); waldenstrom's Macroglobulinemia (Waldenstrom's macrolobalinemia); lymphomas, including histiocytic and T-cell lymphomas, B-cell lymphomas, including hodgkin's lymphoma and non-hodgkin's lymphoma, such as low grade/follicular non-hodgkin's lymphoma (NHL), cellular lymphoma (FCC), Mantle Cell Lymphoma (MCL), Diffuse Large Cell Lymphoma (DLCL), Small Lymphocytic (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, large mass (bulk disease) NHL; solid tumors, including ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular cancer (liver cancer, hepatoma), gallbladder cancer, bile duct cancer, esophageal cancer, renal cell cancer, thyroid cancer, head and neck squamous cell cancer (head and neck cancer), testicular cancer, endocrine adenocarcinoma, adrenal cancer, pituitary cancer, skin cancer, soft tissue cancer, vascular cancer, brain cancer, neural cancer, eye cancer, meningeal cancer, oropharyngeal cancer, hypopharynx cancer, cervical cancer, and uterine cancer, glioblastoma, medulloblastoma (medulloblastoma), astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, myelodysplastic syndrome, neuroblastoma, prostate cancer, bladder cancer, and sarcomas including, but not limited to, osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma (chrondrosarcoma); and melanoma.

Cancer treatment

As is well known to those of ordinary skill in the art, combination therapy is often employed in the treatment of cancer when a single drug treatment or procedure does not adequately treat or cure the disease or condition. Conventional cancer therapy often involves surgery, radiation therapy, the co-administration of cytotoxic drugs to achieve additive and synergistic effects, and combinations of any or all of these approaches. Particularly useful chemotherapy in combination with biotherapy employs drugs that act through different mechanisms of action, thereby increasing cancer cell control or killing, increasing the ability of the immune system to control cancer cell growth, reducing the potential for drug resistance during treatment, and minimizing potential overlapping toxicities by allowing the use of reduced doses of each drug.

Classes of anti-cancer, anti-tumor, and anti-Neoplastic agents useful in combination therapies encompassed by The methods of The present invention are disclosed in, for example, Goodman & Gilman's The Pharmacological Basis of Therapeutics [ Goodman and Gilman's pharmacology base of Therapeutics ], twelfth edition (2010) edited by L.L.Brunton, B.A.Chabner and B.C.Knollmann, section VIII, "Chemotherapy of Neoplastic Diseases ]", chapters 60-63, page 1665 1770, Megracil group, New York, and include, for example, anthracyclines, platinoids, taxol, topoisomerase inhibitors, anti-metabolites, anti-tumor antibiotics, mitotic inhibitors, and alkylating agents, natural products, various miscellaneous agents, hormones and antagonists, targeted drugs, monoclonal antibodies, and other protein Therapeutics.

In addition to the foregoing, the methods of the present disclosure are related to the treatment of cancer indications, and further include treating a patient via surgery, radiation, and/or administering to a patient in need thereof an effective amount of a small chemical molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic agent conventionally used or currently being developed to treat a neoplastic disorder. This includes antibodies and antigen-binding fragments thereof, cytokines, antisense oligonucleotides, sirnas, and mirnas other than those disclosed herein.

The methods of treatment disclosed and claimed herein include the use of the herein disclosed antibodies alone, and/or in combination with each other, and/or in combination with the presently disclosed antigen-binding fragments thereof that bind CD47, and/or also in combination with competing antibodies that exhibit appropriate biological/therapeutic activity, e.g., all possible combinations of these antibody compounds to achieve maximum therapeutic efficacy.

In addition, the treatment methods of the invention also encompass the use of these antibodies, antigen-binding fragments thereof, competitive antibodies and combinations thereof, in further combination with: (1) any one or more antineoplastic therapeutic treatments selected from the group consisting of surgery, radiation, an antineoplastic, an anticancer agent, and combinations of any of these; or (2) any one or more of an anti-neoplastic agent; or (3) any equivalent of (1) or (2) in one or more suitable combinations as would be apparent to one of ordinary skill in the art, in order to achieve the desired therapeutic effect for a particular indication.

The antibodies and small molecule drugs that increase the immune response to cancer BY modulating the co-stimulatory or inhibitory interaction of the T cell response to tumor antigens (including inhibitors of immune checkpoint and modulators of co-stimulatory molecules) are also of particular interest in the context of the combined therapeutic methods encompassed herein, and include, but are not limited to, other anti-CD antibodies, therapeutic agents that bind to CD proteins, such as antibodies or small molecules that bind to CD and prevent interaction between CD and SIRP, such that cancer cells are cleared via phagocytosis, therapeutic agents that bind to CD proteins, such as antibodies, chemical small molecules or biopharmaceutical combinations directed against one or more additional cellular targets, including, but not limited to, CD (differentiation cluster 70), CD200(OX-2 membrane glycoprotein, cluster 200), CD 100 (differentiation cluster 154, CD40, CD ligand, cluster 40 ligand, CD223 (lymphocyte activation gene cluster 3, tmEGF, CD cluster), CD-TNF-7 receptor (TNF-7), TNF-7-TNF-7 receptor.

(Yipriomama; Bezishi Meishibao) is an example of an approved anti-CTLA-4 antibody.

(pembrolizumab; merck) and

(nivolumab; Beshizubao corporation, Behcet.) is an example of an approved anti-PD-1 antibody.

(Attributab; Roche) is an example of an approved anti-PD-L1 antibody.

(Avermezumab, merck, feverine and Lilly (Eli Lilly)) is an example of an approved anti-PD-L1 antibody.

(Duvacizumab; medical immunization/Aslicon) is an example of an approved monoclonal antibody that blocks the interaction of programmed cell death ligand 1(PD-L1) with PD-1 and CD80(B7.1) molecules.

Examples of the invention

Example 1

Amino acid sequence

Light chain CDR

Heavy chain CDR

Murine light chain variable domains

>Vx4murL01

DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

(SEQ ID NO:41)。

>Vx4murL02

DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIK

(SEQ ID NO:42)。

>Vx8murL03

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIK(SEQ ID NO:46)。

>Vx9murL04

DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKVEIK(SEQ ID NO:50)

Murine heavy chain variable domains

>Vx4murH01

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSS(SEQ ID NO:21)。

>Vx4mur-H02

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTLVTVSS(SEQ ID NO:22)。

>Vx8murH03

EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSVTVSS(SEQ ID NO:28)。

>Vx9murH04

QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGGRVGLGYWGHGSSVTVSS(SEQ ID NO:35)

Human light chain variable domains

>Vx4humL01

DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIK(SEQ ID NO:43)

>Vx4humL02

DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK(SEQ ID NO:44)

>Vx4humL03

DIVMTQSPDSLAVSLGERATINCRSRQSIVHTNGNTYLGWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIK

(SEQ ID NO:45)

>Vx8humL04

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIK(SEQ ID NO:47)。

>Vx8humL05

DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK(SEQ ID NO:48)。

>Vx8humL06

DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQGTKLEIK(SEQ ID NO:49)

>Vx9humL07

DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK(SEQ ID NO:51)。

>Vx9humL08

DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIK

(SEQ ID NO:52)。

Human heavy chain variable domains

>Vx4humH01

QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATLVTVSS(SEQ ID NO:23)。

>Vx4humH02

QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATLVTVSS(SEQ ID NO:24)。

>Vx4humH03

EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYVIHWVQQAPGKGLEWMGYIYPYNDGILYNEKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYYVPDYWGQGTTVTVSS(SEQ ID NO:25)

>Vx4humH04

EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTTVTVSS(SEQ ID NO:26)

>Vx4humH05

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGTTVTVSS(SEQ ID NO:27)

>Vx8humH06

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS(SEQ ID NO:29)。

>Vx8humH07

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS(SEQ ID NO:30)。

>Vx8humH08

EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSS(SEQ ID NO:31)。

>Vx8humH09

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS(SEQ ID NO:32)。

>Vx8humH10

EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTLVTVSS(SEQ ID NO:33)。

>Vx8humH11

QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQATLVTVSS(SEQ ID NO:34)

>Vx9humH12

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS(SEQ ID NO:36)。

>Vx9humH13

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS(SEQ ID NO:37)。

>Vx9humH14

EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSS(SEQ ID NO:38)。

>Vx9humH15

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS(SEQ ID NO:39)。

>Vx9humH16

EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSS(SEQ ID NO:40)。

Human IgG-Fc

Human Fc IgG1

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:53)。

Human Fc IgG1-N297Q

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:54)。

Human Fc-IgG2

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:56)。

Human Fc-IgG3

ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK(SEQ ID NO:57)

Human Fc-IgG4

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:58)。

Human Fc-IgG 4S228P

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:59)。

Human Fc-IgG4PE

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:60)

Human Fc-IgG4 PE'

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG(SEQ ID NO:101)

Human kappa LC

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:61)。

Rat Fc-IgG2c

ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK(SEQ ID NO:62)。

Rat kappa LC

RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSSPVVKSFNRNEC(SEQ ID NO:63)。

Rabbit IgG-Fc

Rabbit IgG

GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:64)。

Rabbit kappa LC

RDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC(SEQ ID NO:65)。

>CD47

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE(SEQ IDNO:66)。

Chimeric and human light chains

Vx4murL01 full Length

DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:67)。

Vx4murL01 full Length

DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:68)。

(> Vx4humL01 full-length LC

DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:69)。

(> Vx8humL03 full-length LC

DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:70)。

(> Vx9humL02 full-length LC

DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:71)。

(> Vx8humL02 full-length LC

DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:72)。

(> Vx4humL02 full-length LC

DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:73)。

(> Vx9humL07 full-length LC

DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:74)。

(> Vx8humL01 full-length LC

DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:75)。

> Vx8murL03 full-length LC

DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:76)。

Vx9mur _ L04 full-length LC

DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:77)。

Chimeric and human heavy chains

Vx4murH01 full-length HC

EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:78)。

(> Vx4humH01 full length HC)

QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:79)。

(> Vx8humH11 full length HC)

QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:80)。

Vx9humH12 full-length HC

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:81)。

Vx9humH14 full-length HC

EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:82)。

Vx9humH15 full-length HC

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:83)。

(> Vx4humH02 full length HC)

QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:84)。

Vx9humH13 full-length HC

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:85)。

(> Vx8humH10 full length HC)

EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:86)。

(> Vx4humH04 full length HC)

EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:87)。

(> Vx4humH05 full length HC)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:88)。

Vx9humH16 full-length HC

EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:89)。

(> Vx8humH06 full length HC)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:90)。

(> Vx8humH07 full length HC)

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:91)。

(> Vx8humH08 full length HC)

EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:92)。

(> Vx8humH09 full length HC)

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:93)。

(> Vx8humH06 full length HC)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:94)。

Vx8mur-H03 full-length HC

EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:95)。

Vx9mur-H04 full-length HC

QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGGRVGLGYWGHGSSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:96)。

(> Vx8humH06 full length HC)

QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:97)。

(> Vx8humH07 full length HC)

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:98)。

(> Vx8humH08 full length HC)

EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:99)。

(> Vx8humH09 full length HC)

QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:100)。

Example 2

Production of CD47 antibody

Chimeric antibodies disclosed herein comprise mouse heavy and light chain variable domains in combination with human kappa or human Fc IgG1, IgG1-N297Q, IgG2, IgG4, IgG4S228P, and IgG4PE constant domains, respectively. These were designed to bind to a secretion signal and cloned into a mammalian expression system and transferred into CHO cells to generate chimeric (murine-human) antibodies. Chimeric variants were expressed as full-length IgG molecules, secreted into the culture medium and purified using protein a.

Various methods for humanizing antibodies are well known to those of ordinary skill in the art. As used herein, one such method has been previously described (labeling and Using Antibodies a Practical Handbook [ Making and Using Antibodies, a Handbook of this utility ], second edition, edition Matthey R. Kase, Chapter 15: Humanization of Antibodies [ Chapter 15: Humanization of Antibodies ], Juan carbohydrates Almagro et al, CRC Press 2013 ]). As such, the humanized antibodies disclosed herein comprise a framework derived from a human genome. This series encompasses the diversity found in human germline sequences, resulting in functionally expressed antibodies in vivo. Complementarity Determining Regions (CDRs) of the light and heavy chain variable regions of murine and chimeric (murine-human) are described herein and determined by following the generally accepted rules disclosed below: "Protein Sequence and Structure Analysis of Antibody Variable Domains" in the following: antibody Engineering Lab Manual, edited by S.Duebel and R.Kontermann, Schpringer Press (Springer-Verlag), Heidelberg (2001). Human light chain variable domains were then designed. The humanized variable domains were then combined with secretion signals and human κ and human Fc IgG1, IgG1-N297Q, IgG2, IgG3, IgG4S228P, and IgG4PE constant domains, cloned into mammalian expression systems, and transferred into CHO cells to generate humanized mabs. Humanized variants were expressed as full-length IgG molecules, secreted into the culture medium and purified using protein a.

The aglycosylated form (IgG1-N297Q) was created by site-directed mutagenesis at heavy chain position 297 to change asparagine to glutamine (human Fc IgG1-N297Q, SEQ ID NO: 54). IgG4 variants were created by site-directed mutagenesis at position 228 to change serine to proline, thereby preventing Fab arm exchange in vitro. IgG4 double mutants were created by site-directed mutagenesis at positions 228 (serine to proline) and 235 (leucine to glutamate) to prevent Fab arm exchange and further reduce Fc effector function. IgG2, IgG3, IgG4S228P, and IgG4PE isotypes were constructed by cloning the heavy chain variable domain in frame with human IgG2, IgG3, IgG4S228P, and IgG4PE constant domains (human Fc-IgG2, SEQ ID NO: 56; human Fc-IgG3, SEQ ID NO: 57; human Fc-IgG 4S228P, SEQ ID NO: 59; and human Fc-IgG4PE, SEQ ID NO: 60).

Example 3

Binding of CD47 monoclonal antibody (mAb)

The binding of the chimeric (murine-human) and humanized antibodies of the present disclosure was determined by ELISA using OV10 cells transfected with human CD47(OV10 hCD47) or using freshly isolated human red blood cells (hRBC) displaying CD47 on their surface (Kamel et al (2010) blood.

The binding activity of VLX4, VLX8 and VLX9 chimeric (xi) and humanized mabs was determined using a cell-based ELISA assay using human OV10hCD47 cells expressing cell surface human CD 47. OV10hCD47 cells were grown in IMDM medium containing 10% heat-inactivated fetal bovine serum (BioWest, Inc.; S01520). The day before the measurement, 3X 10⁴Individual cells were seeded in 96-well cell binding plates (Corning) #3300, VWR # 66025-. Cells were washed, different concentrations of purified antibody were added to IMDM, and 95% O at 37 ℃₂/5％CO₂And incubated for 1 hour. The cells were then washed with medium and incubated for an additional hour at 37 ℃ with a dilution of 1/2500 of HRP-labeled secondary anti-human antibody (Promega) in medium. The cells were washed three times with PBS and the

peroxidase substrate

3,3',5,5' -tetramethylbenzidine (Sigma; Cat. No. T4444) was added. The reaction was stopped by adding HCl to 0.7N and read using an Infinite M200 plate of Diken (Tecan) typeThe absorbance was measured at 450nM of counter. The apparent binding affinity of these clones to human OV10hCD47 cells was determined by non-linear fitting (Prism GraphPad software).

The binding activity of the chimeric and humanized VLX4, VLX8, and VLX9 mabs to human CD47 on hrbcs was also determined using flow cytometry. Blood was obtained from normal volunteers and RBC were washed 3 times with phosphate buffered saline pH 7.2 (PBS + E) containing 2.5mM EDTA. hRBC were incubated with varying concentrations of chimeric or humanized antibody in PBS + E for 60 minutes at 37 ℃. The cells were then washed with cold PBS + E and incubated with FITC-labeled donkey anti-human antibody in PBS + E (Jackson Immuno Research Labs, West Grove, Pa., catalog number 709-. Cells were washed with PBS + E, antibody binding was analyzed using a C6 Accuri flow cytometer (bicdy (Becton Dickinson)), and apparent binding affinities were determined by nonlinear fitting (Prism GraphPad software) for median fluorescence intensity at various antibody concentrations.

All VLX4 chimeric (murine-human) mabs bound to human OV10hCD47 tumor cells with apparent affinities in the picomolar (pM) range (table 1).

Similarly, the humanized VLX4 mAb bound to human OV10hCD47 tumor cells (table 2) in a concentration-dependent manner (fig. 1A and 1B) with an apparent binding affinity ranging from picomolar to low nanomolar.

All chimeric VLX4 mabs bound to human RBCs with apparent Kd values in the picomolar range and these values were similar to K obtained by ELISA for OV10hCD47 tumor cells_dValues (table 1).

Humanized VLX4 mabs VLX4hum _01IgG 1N 297Q, VLX4hum _02IgG 1N 297Q, VLX4hum _01IgG4PE, VLX4hum _02IgG4PE, VLX4hum _12 IgG4PE, and VLX4hum _13IgG4PE bound to human RBCs with Kd values similar to those obtained for OV10hCD47 tumor cells, while VLX4hum _06 IgG4PE and VLX4hum _07IgG4PE showed reduced binding to hrbcs (fig. 2A, fig. 2B, and table 2). The differential binding of the humanized mAb to tumor cells and RBCs was unexpected when the VLX4IgG 4PE chimeric mAb bound tumor and RBC CD47 with similar apparent Kd values (table 1).

As shown in table 1, all VLX8 chimeric mabs bound to human OV10hCD47 tumor cells in a concentration-dependent manner with apparent affinity in the picomolar (pM) range.

Similarly, the humanized VLX8 mAb bound to human OV10hCD47 tumor cells (table 2) with apparent affinity in the picomolar range in a concentration-dependent manner (fig. 3A and 3B).

Apparent K in picomolar range for all VLX8 chimeric mAbs_dValues bound to human hRBCs and these values were similar to the apparent K obtained by ELISA for OV10hCD47 tumor cells_dValues (table 1).

VLX8 humanized mabs VLX8hum _01IgG4PE, VLX8hum _02IgG4PE, VLX8hum _03 IgG4PE, VLX8hum _04 IgG4PE, VLX8hum _05 IgG4PE, and VLX8hum _06 IgG4PE, VLX8hum _07IgG4PE, VLX8hum _08IgG4PE, VLX8hum _09 IgG4PE, VLX8hum _11IgG4PE, VLX8hum _06IgG2, VLX8hum _07 IgG2, VLX8hum _08, and VLX8hum _09 IgG 2IgG2 bound to humans with Kd values similar to those obtained for OV10hCD47 tumor cells, while VLX8hum _10 IgG4 RBC 4PE showed reduced binding to RBC 4A, fig. 4B (table 4B). Unexpectedly, the differential binding of the humanized mAb to tumor cells and RBCs was that the VLX8IgG4PE chimeric mAb bound tumor and RBC CD47 with similar apparent Kd values (table 1).

Table 1 shows the apparent binding affinity of the VLX9 chimeric mAb to human OV10hCD47 cells and to human RBCs. All chimeric mabs bound to OV10hCD47 tumor cells with an apparent binding constant in the picomolar range. Similarly, the humanized VLX9mAb bound to human OV10hCD47 tumor cells (table 2) with apparent affinity in the picomolar to nanomolar range in a concentration-dependent manner (fig. 5A and 5B).

Apparent K in picomolar range for all VLX9 chimeric mAbs_dValues bound to human hRBCs and these values were similar to the apparent K obtained by ELISA for OV10hCD47 tumor cells_dValues (table 1). Compared to the chimeric mabs, VLX9 humanized mAbVLX9hum _01 IgG2, VLX9hum _02IgG2, and VLX9hum _07 IgG2 showed reduced binding to human RBCs (fig. 7, table 2). By comparison, humanized mAbs VLX9hum _03 IgG2, VLX9hum _04 IgG2, VLX9hum _05 IgG2, VLX9hum _06IgG2, VLX9hum _08IgG2, VLX9hum _09 IgG2, and VLX9hum _10 IgG2 did not exhibit measurable binding to RBCs up to 5,000pM (Table 2). This differential binding of the humanized mAb to tumor cells and RBCs was unexpected when both VLX9 IgG2 chimeric mabs bound tumor and RBC CD47 with similar apparent Kd values (table 1).

Specific binding of the CD47 humanized mAb was demonstrated using Jurkat wild type and Jurkat CD47 knock-out (KO) cells. Jurkat wild type and Jurkat CD47KO cells were grown in RPMI medium containing 10% heat-inactivated fetal bovine serum (BioWest, Inc.; S01520). The cells were washed and 1X 10 cells were washed⁴The individual cells were resuspended in culture medium and incubated at 37 ℃ in 5% CO₂Incubated with various antibody concentrations for 1 hour. The cells were then washed twice with 1 XPBS and then at 37 ℃ in 5% CO₂In a secondary antibody (goat anti-human IgG (H + L) FITC-labeled, Jackson laboratory, 109-. The cells were then washed twice with 1 × PBS and resuspended in 1 × PBS. Median fluorescence intensity was determined by flow cytometry and apparent binding affinity was determined by nonlinear fitting (Prism GraphPad software).

As shown in fig. 6, VLX4hum _07IgG4PE (fig. 6A) and VLX9hum _09 IgG2 (fig. 6B) bound to Jurkat cells expressing CD47, while no binding was observed to Jurkat CD47KO cells.

Table 1:binding of VLX4, VLX8, and VLX9 chimeric (xi) mabs to OV10hCD47 cells and human blood red blood cells (hRBC).

Table 2.Humanized mabs to VLX4, VLX8 and VLX9 and human OV10 Binding of hCD47 to human blood red blood cells (hrbcs).

MB-minimal binding; no measurable binding was detected up to 5,000pM at mAb concentration.

Decreased RBC binding.

Decreased hemagglutination.

Cross species binding of humanized VLX4, VLX8 and VLX9 mabs was determined using flow cytometry. Mouse, rat, rabbit or cynomolgus RBCs were incubated with varying concentrations of humanized antibody in phosphate buffered saline (pH 7.2), 2.5mM EDTA (PBS + E) solution for 60 minutes at 37 ℃. The cells were then washed with cold PBS + E and incubated with FITC-labeled donkey anti-human antibody in PBS + E (Jackson Immunity research laboratory, Sjogrev, Pa.; Cat. 709-096-149) for an additional hour on ice. Cells were washed with PBS + E and analyzed for antibody binding using a C6 Accuri flow cytometer (bidi).

Table 3 shows the apparent binding affinity of humanized VLX4 and VLX8 mabs to RBCs from mice, rats, and cynomolgus monkeys as determined by non-linear fitting of intermediate fluorescence intensities at different antibody concentrations (Prism GraphPad software). This data indicates that humanized VLX4 and VLX8 mAb bind mouse, rat, rabbit (data not shown) and cynomolgus RBCs with apparent Kd values in the picomolar to nanomolar range.

Table 3.Binding of VLX4 and VLX8 humanized mabs to mouse, rat and cynomolgus RBCs.

Example 4

Humanized anti-CD 47 determination by surface plasmon resonance Binding of mAbs

The binding of soluble anti-CD 47mAb to recombinant human His-CD47 was measured in vitro by surface plasmon resonance on Biacore 2000. Anti-human IgG (GE medical life sciences) is an amine coupled to CM5 chips on

flow cells

1 and 2. Will be in HBS-EP⁺Humanized mAb VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _08 IgG2, or VLX9hum _03 IgG2 diluted in running buffer (pH 7.2) captured to streamThe moving pool 2. Used in HBS-EP⁺The multi-cycle kinetics were determined from 0 to 1000nM His-tagged human CD47(Acro Biosystems) diluted in running buffer (pH 7.2) with a contact time of 180 seconds and a dissociation time of 300 seconds. The method comprises the following steps of 1:1 binding model kinetic analysis of binding curves was performed. The on-rate, off-rate and off-constant for VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _08 IgG2 and VLX9hum _03 IgG2 are shown in table 4.

Table 4.Humanized mabs and human recombinants determined by surface plasmon resonance at pH 7.2 VLX4, VLX8 and VLX9 His-CD47 binding.

	k_a	k_d	K_D(nM)
				VLX4hum_07 IgG4PE	1.7e⁵	8.7e^-4	5.1
VLX8hum_11 IgG4PE	6.8e⁵	7.9e^-4	1.2
				VLX9_08 IgG2	7.6e⁴	6.5e^-4	8.6
VLX9_03 IgG2	6.5e⁴	7.3e^-4	11.1

Example 5

Differential binding of anti-CD 47mAb

Some of the soluble CD47 antibodies described herein have been shown to differentially bind to normal cells. This additional property of selective binding is expected to have advantages over mabs that bind normal and tumor cells with the same affinity. anti-CD 47 mabs with such reduced binding have not been described.

Binding of soluble anti-CD 47mAb was measured in vitro. Human Aortic Endothelial Cells (HAEC), skeletal muscle cells (SkMC), human pulmonary microvascular endothelial cells (HMVEC-L), Renal Tubular Epithelial Cells (RTEC), CD3 using flow cytometry-based binding assays⁺The binding activity of VLX4, VLX8, and VLX9 humanized mabs was determined by cells or Peripheral Blood Mononuclear Cells (PBMCs). HAEC, SkMC, HMVEC-L and RTEC cells were purchased from Longsha (Lonza) and cultured according to the manufacturer's recommendations. Adherent cells were removed from the flask with accutase, resuspended in recommended medium, and incubated at 37 ℃ with 5% CO₂The lower part is 1 × 10⁴Individual cells were incubated with various antibody concentrations for 1 hour. For nonadherent cells, 1X 10⁴The individual cells were resuspended in the recommended medium and 5% CO at 37 deg.C₂Incubate with various antibody concentrations for 1 hour. The cells were then washed twice with 1 XPBS and then 5% CO at 37 ℃₂Resuspension was performed in a secondary antibody (goat anti-human IgG (H + L) -FITC, Jackson laboratory, 109-.

PBMCs were isolated by ficoll gradient and incubated with FcR blocking reagent (Miltenyi Biotec) at 4 ℃ for 10 min as recommended by the manufacturer immediately prior to addition of various concentrations of antibody diluted in PBS. CD3 cells were detected using an Allophycocyanin (APC) -labeled anti-CD 3 antibody (BD biosciences) added simultaneously with a FITC-labeled goat anti-human IgG (H + L) antibody. Cells were washed twice with 1 × PBS and antibody binding was assessed by flow cytometry analysis.

As shown in figure 8A, VLX4 and VLX8 humanized mabs bound to HAEC cells, whereas VLX9 humanized mAb had reduced or minimal binding to HAEC cells compared to tumor cells (table 5). The VLX9 humanized mAb also showed reduced binding to SkMC cells (fig. 8B), reduced or minimal binding to HMVEC-L cells (fig. 8C), reduced binding to RPTEC cells (fig. 8D) compared to tumor cell binding (table 5). Compared to tumor cells, humanized mAb VLX9 was also observed with CD3⁺Decreased binding of cells (fig. 8E) and PBMCs (fig. 8F) (table 5). This selective binding confers additional desirable antibody properties and potential therapeutic benefits in cancer treatment.

Table 5.Binding of VLX4, VLX8, and VLX9 humanized mabs to normal cells.

MB-minimal binding, no measurable binding was detected up to 5,000pm at mAb concentration.

-decreased binding.

Example 6

pH-dependent and independent binding of humanized anti-CD 47mAb

Some of the soluble anti-CD 47 mabs described herein have been shown to bind tumor cells with greater affinity at acidic pH than at physiological pH. This additional property is expected to have advantages over mabs that bind CD47 with similar affinity at both acidic and physiological pH, in part due to the acidic nature of the tumor microenvironment (Tannock and Rotin, Cancer research 1989; Song et al Cancer Drug Discovery and Development 2006; Chen and Pagel, Advan radio 2015 advanced radiology).

The binding of soluble anti-CD 47mAb to immobilized recombinant human CD47 and human CD47 expressed on cells was measured in vitro. For in vitro binding of recombinant CD47, His-CD47 (Acrobiosystems) was adsorbed onto high binding microtiter plates overnight at 4 ℃. The wells were washed and different concentrations of anti-CD 47mAb were added to the wells in buffer at pH 6 or pH 8 for 1 hour. The wells were washed at pH 6 or pH 8 and then incubated with HRP-labeled secondary antibody for 1 hour, followed by addition of peroxidase substrate. Apparent affinities were calculated using a non-linear fitting model (Graphpad Prism).

To analyze pH-dependent binding by surface plasmon resonance using Biacore 2000, anti-human IgG (GE medical life science) was an amine coupled to CM5 chips on

flow cells

1 and 2. Fc-tagged human CD47 (Acrobiosystems) was placed in PBS-EP⁺Diluted in running buffer (pH 7.5, 6.5 or 6.0) and captured onto flow cell 2. Used in PBS-EP⁺The multi-cycle kinetics were determined for 0 to 100nM VLX8hum _11Fab or VLX9hum _08Fab diluted in running buffer (pH 7.5, 6.5 or 6.0) with a contact time of 180 seconds and a dissociation time of 300 seconds. Kinetic analysis of binding curves was performed using a 1:1 binding model.

For in vitro binding of CD47 expressing cells, Jurkat cells were grown in RPMI medium containing 10% heat-inactivated fetal bovine serum (BioWest, Inc.; S01520). The cells were washed and 1X 10 cells were washed⁴Individual cells were resuspended in PBS supplemented with 2% FBS at pH 7.4 or 6.5 and incubated with various antibody concentrations for 1 hour at 37 ℃. The cells were then washed twice and resuspended for 1H with a 1:1000 secondary antibody (goat anti-human IgG labeled with Alexa488 (H + L), Jackson Immunoresearch) at 37 deg.C, pH 6 or pH 8. The cells were then washed twice and the median fluorescence intensity was determined by flow cytometry. Apparent binding affinity was determined by non-linear fitting (Prism GraphPad software).

As shown in fig. 9A and 9B, soluble VLX9 humanized mabs (VLX9hum _09 IgG2 and VLX9hum _04 IgG2) bound to His-CD47 with greater affinity at more acidic pH 6.0 than at pH 8.0. Neither VLX4hum _07IgG4PE (fig. 9C) nor VLX8hum _10 IgG4PE (fig. 9D) displayed pH-dependent binding. In addition, murine VLX9 antibody and human Fc-containing VLX9 chimeric antibodies from isotypes IgG1, IgG2, and IgG4PE did not exhibit pH dependence (table 6), while VLX9hum _04, which is IgG1, IgG2, or IgG4PE, exhibited greater binding to His-CD47 at acidic pH (table 7). The apparent binding affinity of additional humanized mabs to recombinant human CD47 is shown in table 8. All humanized VLX9 mabs showed pH-dependent binding, whereas VLX4 and VLX8 humanized mabs did not show such binding. To determine the effect of pH on the binding rate, dissociation rate and dissociation constant, Biacore analysis was performed on the humanized mabs VLX8hum _11Fab fragment and VLX9hum _08Fab at pH 6, pH 6.5 and pH 7.5. VLX9hum _08Fab exhibited pH-dependent binding, which increased with decreasing pH, whereas VLX8hum _11Fab did not exhibit such binding. The association rate, dissociation rate and dissociation constant of VLX8hum _11Fab and VLX9hum _08Fab are shown in table 9. Table 10 shows the pH-dependent binding exhibited by VLX9hum _04 IgG2 to CD47 expressed on Jurkat cells. VLX4hum — 07IgG4PE showed no pH-dependent binding. This pH dependence of the VLX9 humanized mAb confers additional desirable antibody properties and therapeutic benefits in cancer treatment.

Table 6.Binding of murine VLX9 and mouse-human chimeric VLX9 to CD47 is not pH dependent

Table 7.The VLX9hum _04 humanized mAb binds CD47 in a pH-dependent manner and binds not isoform specific

Table 8.pH-dependent and independent binding of VLX4, VLX8, and VLX9 humanized mabs.

	KD(pM)pH 6	KD(pM)pH 8
			VLX9hum_03 IgG2	48	>33,000
VLX9hum_04 IgG2	43	>33,000
			VLX9hum_06 IgG2	61	>33,000
VLX9hum_08 IgG2	65	>33,000
			VLX9hum_09 IgG2	138	>33,000
VLX4hum_07 IgG4PE	63	92
			VLX4hum_01 IgG4PE	47	75
VLX8hum_10 IgG4PE	52	79
			VLX8hum_11 IgG4PE	64	92

Table 9.pH-dependent and independent binding of VLX8hum _11Fab and VLX9hum _08Fab to recombinant human CD47

Table 10.pH-dependent and independent binding of VLX4 and VLX9 humanized mAbs to Jurkat cells

Example 7

CD47 antibody blocks CD47/SIRP α binding

To evaluate the effect of humanized CD47mAb on the binding of CD47 to SIRP α in vitro, a method was employed that used the binding of a fluorescently labeled SIRP α -Fc fusion protein to Jurkat cells expressing CD 47.

Alexa was used according to the manufacturer's instructions

Antibody labeling kit (Invitrogen catalog number A20186) for labeling SIRP α -Fc fusion protein (Andy Bio Inc. (R)&D Systems), directory number 4546-SA). At 37 ℃, 1.5X 10⁶Individual Jurkat cells were incubated for 30 minutes with RPMI containing 10% medium or humanized mAb (5 μ g/ml) as a human control antibody in medium alone, an equal volume of fluorescently labeled SIRP α -Fc fusion protein was added and incubated for an additional 30 minutes at 37 ℃, the cells were washed once with PBS and the amount of labeled SIRP α -Fc binding to Jurkat cells was analyzed by flow cytometry.

As shown in figure 10, humanized VLX4, VLX8, and VLX9 mabs (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _10 IgG4PE, VLX8hum _11IgG4PE, VLX9hum _03 IgG2, VLX9hum _06IgG2, and VLX9hum _08 IgG2) blocked the interaction of CD47 expressed on Jurkat cells with soluble SIPR α, while the human control antibody (does not bind CD47) or media alone did not block the CD47/SIRP α interaction.

Example 8

CD47 antibodies increase phagocytosis

To evaluate the effect of chimeric (murine-human) and humanized VLX4, VLX8, and VLX9 CD47 mabs on phagocytosis of tumor cells by macrophages in vitro, the following method using flow cytometry was employed (Willingham et al (2012) Proc natl Acad Sci USA [ journal of the national academy of sciences USA ]109(17):6662-7 and Tseng et al (2013) Proc natl Acad Sci U S a [ journal of the national academy of sciences ]110(27): 11103-8).

Human-derived macrophages were derived from the leukocyte depletion of healthy human peripheral blood and incubated in AIM-V medium (Life Technologies) for 7-10 days. For in vitro phagocytosis assays, macrophages were measured at 1 × 10⁴The concentration of individual cells/well was re-seeded in 100ul of AIM-V medium in 96-well plates and allowed to grow adherently for 24 hours. Once effector macrophages are attached to the dish, target human cancer cells (Jurkat) are labeled with 1. mu.M 5(6) -carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and plated in 1ml AIM-V medium 5X 10⁴Individual cell concentrations (5:1 target to effector ratio) were added to macrophage cultures. VLX4, VLX8, and VLX9 CD47mAb (1 μ g/ml) were added at the time of mixing target and effector cells and allowed to incubate at 37 ℃ for 2-3 hours. After 2-3 hours, all non-phagocytized cells were removed and the remaining cells were washed three times with phosphate buffered saline (PBS; Sigma Aldrich). The cells were then trypsinized, collected in a microcentrifuge tube, and incubated in 100ng of Allophycocyanin (APC) -labeled CD14 antibody (BD biosciences) for 30 minutes, washed once, and analyzed for CD14 by flow cytometry (Accuri C6; BD biosciences)⁺CellsThese cells are also CFSE indicating complete phagocytosis⁺。

As shown in FIG. 11, the VLX4 chimeric mAbs VLX4IgG 1 xi, VLX4IgG 1N 297Q xi, VLX4IgG 4PExi, and VLX4IgG 4S228P xi increase human macrophage phagocytosis of Jurkat cells by blocking CD47/SIRP α interaction.

Similarly, as shown in FIGS. 12A and 12B, the humanized mAbs VLX4hum _01 IgG1, VLX4hum _01IgG4PE, VLX4hum _06 IgG4PE, VLX4hum _07IgG4PE, VLX4hum _12 IgG4PE, and VLX4hum _13IgG4PE increase human macrophage phagocytosis of Jurkat cells by blocking CD47/SIRP α interactions.

As shown in FIG. 13A, the VLX8 chimeric mAbs VLX8IgG 1N 297Q xi and VLX8IgG4PE xi increase human macrophage phagocytosis of Jurkat cells by blocking the CD47/SIRP α interaction.

Similarly, as shown in fig. 13B, the humanized mabs VLX8hum _01IgG4PE, VLX8hum _03 IgG4PE, VLX8hum _07IgG4PE, VLX8hum _08IgG4PE, and VLX8hum _09 IgG4PE and the chimeric mAb VLX8IgG4PE xi increase phagocytosis of Jurkat cells by human macrophages by blocking CD47/SIRP α interactions.

Similarly, as shown in FIG. 14B, all of the humanized VLX9 IgG2 mAbs (VLX9hum _01 to _10 IgG2) increased phagocytosis of Jurkat cells by blocking CD47/SIRP α interactions.

Example 9

Induction of cell death by soluble CD47 antibody

This additional property of selective toxicity against cancer cells is expected to have an advantage over mabs that block only the binding of SIRP α to CD 47.

In vitroMeasurement of the induction of cell death by soluble anti-CD 47mAb (Manna et al (2003) J.Immunol. [ J.Immunol. ]]107(7) 3544-53, Kikuchi et al Biochem Biophys Res. Commun [ Biochemical and biophysical research communication ]]315:912-8,2004, Pettersen et al J.Immuno. [ J.Immunol. ]]162 7031-],64:1026-1036,2004). For in vitro cell death assays, 1X 10 cells were incubated at 37 deg.C⁵Individual transformed human T cells (Jurkat cells) were incubated with soluble humanized VLX4, VLX8, and VLX9 CD47mAb (1 μ g/ml) for 24 hours. As cell death occurs, the mitochondrial membrane potential decreases, the inner lobes of the cell membrane invert, exposing Phosphatidylserine (PS), and Propidium Iodide (PI) or 7-amino actinomycin D (7-AAD) can bind to nuclear DNA. To detect these cellular changes, cells were then stained with fluorescently labeled annexin V and PI or 7-amino actinomycin D (7-AAD) (BD biosciences) and signals were detected using an Accuri C6 flow cytometer (BD biosciences). The increase in PS exposure was determined by measuring the percentage increase in annexin V signal and the percentage of dead cells was determined by measuring the percentage increase in PI or 7-AAD signal. Annexin V positivity was observed at the early stage of cell death (annexin V)⁺) Or annexin V positive/7-AAD negative (annexin V)⁺/7-AAD^-) Cells, and annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) The cell is a dead cell. Importantly for therapeutic purposes, these mabs directly induce cell death of tumor cells and do not require supplementation or intervention with other cells (e.g., NK cells, T cells, or macrophages) for killing. Thus, the mechanism is independent of other cells and Fc effector functions. Thus, therapeutic antibodies developed from these mabs can be engineered to reduce Fc effector functions such as ADCC and CDC and thus limit the possibility of side effects common to humanized mabs with intact Fc effector function.

As shown in FIGS. 15A-15F, soluble VLX4 humanized mAb induced increased PS exposure and cell death of Jurkat cells, as by being annexin V⁺(FIGS. 15A and 15D), annexin V⁺/7-AAD^-(FIGS. 15B and 15E), or annexin V⁺/7-AAD⁺(FIG. 15C and FIG. 15F) as measured by% increase in cells. Humanized mabs VLX4hum _01 IgG1, VLX4hum _01IgG4PE, VLX4hum _02 IgG1, VLX4hum _02IgG4PE, VLX4hum _06 IgG4PE, VLX4hum _07IgG4PE, VLX4hum _12 IgG4PE, and VLX4hum _13IgG4PE caused increased PS exposure and cell death. In contrast, the humanized mabs VLX4hum _08IgG4PE and VLX4hum _11IgG4PE did not cause increased PS exposure and cell death of Jurkat cells. Inducing cell death and promoting phagocytosis of susceptible cancer cells confers additionally desirable antibody properties and potential therapeutic benefits in cancer therapy.

As shown in FIGS. 16A-16F, soluble VLX8 chimeric and humanized mAbs induced increased PS exposure and cell death of Jurkat cells, as by being annexin V⁺(FIGS. 16A, 16D), annexin V⁺/7-AAD^-(FIGS. 16B, 16E), or annexin V⁺/7-AAD⁺(FIGS. 16C, 16F) as measured by% of cells. Chimeric mabs VLX8IgG 1N 297Q xi and VLX8IgG 4PExi, and humanized mabs VLX8hum _07IgG4PE and VLX8hum _08IgG4PE induced increased PS exposure and cell death of Jurkat cells. In contrast, the humanized mabs VLX8hum _02IgG4PE and VLX8hum _04 IgG4PE did not cause increased PS exposure and cell death of Jurkat cells. Inducing cell death and promoting phagocytosis of susceptible cancer cells confers additionally desirable antibody properties and potential therapeutic benefits in cancer therapy.

As shown in FIGS. 17A-17F, soluble VLX9 chimeric and humanized antibodies induced increased PS exposure and cell death of Jurkat cells, as by annexin V⁺(FIGS. 17A and 17D), annexin V⁺/7-AAD^-(FIGS. 17B and 17E), or annexin V⁺/7-AAD⁺(FIG. 17C and FIG. 17F) as measured by% of cells. The chimeric VLX9 IgG2xi mAb and the humanized mabs VLX9hum _06IgG2, VLX9hum _07 IgG2, VLX9hum _08 IgG2, and VLX9hum _09 IgG2 induced increased PS exposure and cell death of Jurkat cells. In contrast, the humanized mabs VLX9hum _01 IgG2, VLX9hum _02IgG2, VLX9hum _03 IgG2, VLX9hum _04 IgG2, VLX9hum _05 IgG2, and VLX9hum _010 IgG2 did not cause PS exposure and cell death of Jurkat cellsThe death is increased. Inducing cell death and promoting phagocytosis of susceptible cancer cells confers additionally desirable antibody properties and potential therapeutic benefits in cancer therapy. Importantly, chimeric and humanized mabs that cause cell death of tumor cells do not cause cell death of normal cells.

Example 10

From humanized anti-CD 47 mAb-induced injury-associated molecular Pattern (DAMP) expression and Release, mitochondrial depolarization and cell death

Humanized anti-CD 47mAb causes loss of mitochondrial membrane potential

These experiments demonstrated that the humanized anti-CD 47mAb of the present disclosure exhibited the ability to induce loss of mitochondrial membrane potential in tumor cells as previously described (Manna and Frazier,2014Journal of Immunology 170(7): 3544-3553).

Loss of mitochondrial membrane potential in tumor cells was determined using JC-1 dye (Thermo Co.; catalog No. M34152). Human Raji lymphoma cells (ATCC, Manassas, Va.; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 will be used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells of Raji are plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06 IgG2VLX9hum _08 IgG2, and VLX9hum _03 IgG2) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for mitochondrial membrane potential loss, cells were treated with 1. mu.M of the chemotherapeutic anthracycline mitoxantrone. Cells were incubated at 37 ℃ for 24 hours, then harvested, washed twice with PBS, and incubated with JC-1 dye for 30 minutes as described above, diluted 1:2000 in PBS. After 30 minutes, the cells were washed twice with PBS, resuspended in 100 μ l PBS, and analyzed by flow cytometry (Accuri C6, bidi, franklin lake, nj) for the percentage of cells that changed fluorescence emission from red to green. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPad Prism 6.

Some chimeric or humanized antibodies induce a loss of mitochondrial membrane potential in tumor cells. As shown in figure 18, the percentage of cells with mitochondrial membrane depolarization was significantly increased in all anti-CD 47 mAb-treated cultures compared to isotype control (p < 0.05). This increase in the amount of mitochondrial membrane depolarization indicates that the anti-CD 47 chimeric or humanized antibody induces mitochondrial depolarization leading to cell death in human tumor cells.

Humanized anti-CD 47mAb causes increased cell surface calreticulin expression

These experiments demonstrate that the humanized anti-CD 47 mabs of the present disclosure exhibit the ability to expose the endoplasmic reticulum resident chaperone calreticulin on the surface of tumor cells, for example, as previously described using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Obeid et al (2007) nat. med. [ natural medicine ]13(1): 54-61.

Cell surface exposure of calreticulin was determined using rabbit monoclonal antibodies against calreticulin conjugated to Alexa Fluor 647 (Albam;. Cat ab 196159). Human Raji lymphoma cells (ATCC, Manassas, Va.; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 will be used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵Individual cells/ml RPMI-1640 medium at a density in 96-well tissue culture plates, the medium containingThere were 10% (v/v) heat-inactivated bovine fetal serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, VLX9hum _03 IgG2) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for calreticulin exposure, cells were treated with 1 μ M of the chemotherapeutic anthracycline mitoxantrone. Cells were incubated at 37 ℃ for 24 hours, then harvested, washed twice with PBS, and incubated with anti-calreticulin antibody for 30 minutes as described above, diluted 1:200 in PBS. After 30 minutes, the cells were washed twice with PBS, resuspended in 100 μ l PBS, and analyzed by flow cytometry (Accuri C6, bidi, franklin lake, nj) for mean fluorescence intensity of anti-calreticulin antibody signals and the percentage of cells staining positive for cell surface calreticulin. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPad Prism 6.

As shown in figure 19, humanized antibodies induced pre-apoptotic exposure of calreticulin on the surface of tumor cells. The percentage of calreticulin positive cells was significantly increased in all anti-CD 47mAb treated cultures compared to isotype control (p < 0.05). This increase in calreticulin exposure on the cell surface suggests that some humanized antibodies induce DAMP from tumor cells, which can lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased expression of protein disulfide isomerase 3(PDIA3)

These experiments demonstrate that the humanized anti-CD 47 mabs of the present disclosure exhibit the ability to expose the endoplasmic reticulum resident partner PDIA3 on the surface of tumor cells, e.g., as previously described using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Panaretakis et al (2008) Cell Death & Differentiation [ Cell Death and Differentiation ]15: 1499-1509.

Use with FITC (Albo-antibody)A company; directory number ab183396) conjugated mouse monoclonal antibody against PDIA3 cell surface exposure of PDIA3 was determined. Human Raji lymphoma cells (ATCC, Manassas, Va.; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 will be used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for PDIA3 exposure, cells were treated with 1 μ M of the chemotherapeutic anthracycline mitoxantrone. The cells were incubated for 24 hours at 37 ℃ and then harvested, washed twice with PBS, and incubated with anti-PDIA 3 antibody for 30 minutes as described above, diluted 1:200 in PBS. After 30 minutes, the cells were washed twice with PBS, resuspended in 100 μ l PBS, and analyzed by flow cytometry (Accuri C6, bidi, franklin lake, nj) for mean fluorescence intensity of anti-PDIA 3 antibody signal and the percentage of cells staining positive for cell surface calreticulin. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPad Prism 6.

Some chimeric or humanized antibodies induce pre-apoptotic exposure of PDIA3 on the surface of tumor cells. As shown in figure 20, the percentage of PDIA3 positive cells in all soluble anti-CD 47mAb treated cultures was significantly increased (p <0.05) compared to background obtained with the negative control, humanized isotype-matched antibody. This increase in PDIA3 exposure on the cell surface suggests that some chimeric or humanized antibodies induce DAMP from tumor cells, which can lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased cell surface HSP70 expression

These experiments demonstrate that the humanized anti-CD 47 mabs of the present disclosure exhibit the ability to expose the endoplasmic reticulum resident partner HSP70 on the surface of tumor cells, for example, as previously described using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Fucikova et al (2011) Cancer Research 71(14): 4821-.

Cell surface exposure of HSP70 was determined using a mouse monoclonal antibody directed against HSP70 conjugated to phycoerythrin (ebola; cat # ab 65174). Human Raji lymphoma cells (ATCC, Masnasas, Virginia; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 were used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for HSP70 exposure, lange cells were treated with 1 μ M of the chemotherapeutic anthracycline mitoxantrone. Cells were incubated at 37 ℃ for 24 hours, then harvested, washed twice with PBS, and incubated with anti-HSP 70 antibody for 30 minutes as described above, diluted 1:200 in PBS. After 30 minutes, the cells were washed twice with PBS, resuspended in 100 μ l PBS, and analyzed by flow cytometry (Accuri C6, bidi, franklin lake, nj) for mean fluorescence intensity of anti-HSP 70 antibody signals and the percentage of cells staining positive for cell surface calreticulin. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPad Prism 6.

Some chimeric or humanized antibodies induce a pre-apoptotic exposure of HSP70 on the surface of tumor cells. As shown in figure 21, the percentage of HSP70 positive cells in all anti-CD 47mAb treated cultures was significantly increased (p <0.05) compared to those observed in isotype control treated cultures. This increase in HSP70 exposure on the cell surface suggests that some chimeric or humanized antibodies induce DAMP from tumor cells and may lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased cell surface HSP90 expression

These experiments demonstrate that the humanized anti-CD 47 mabs of the present disclosure expose the endoplasmic reticulum resident partner HSP70 on the surface of tumor cells, e.g., as previously described using chemotherapeutic anthracyclines such as doxorubicin and mitoxantrone, as disclosed by Fucikova et al (2011) Cancer Research [ Cancer Research ]71(14): 4821-4833.

Cell surface exposure of HSP90 was determined using a mouse monoclonal antibody directed against HSP70 conjugated to phycoerythrin (ebola; cat # ab 65174). Human Raji lymphoma cells (ATCC, Masnasas, Virginia; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 were used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 Medium was seeded at a density in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (West)The company gamma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for HSP90 exposure, cells were treated with 1 μ M of the chemotherapeutic anthracycline mitoxantrone. Lange cells were incubated at 37 ℃ for 24 hours, then the cells were harvested, washed twice with PBS, and incubated with anti-HSP 70 antibody for 30 minutes as described above, diluted 1:200 in PBS. After 30 minutes, the cells were washed twice with PBS, resuspended in 100 μ l PBS, and analyzed by flow cytometry (Accuri C6, bidi, franklin lake, nj) for mean fluorescence intensity of anti-HSP 70 antibody signals and the percentage of cells staining positive for cell surface calreticulin. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPad Prism 6.

Some chimeric or humanized antibodies induce a pre-apoptotic exposure of HSP90 on the surface of tumor cells. As shown in figure 22, the percentage of HSP90 positive cells in soluble anti-CD 47mAb treated cultures was significantly increased (p <0.05) compared to the background obtained with the negative control, humanized isotype matched antibody, except VLXhum _06IgG2 and VLX4hum _01IgG4PE (ns, not significant). This increase in HSP90 exposure on the cell surface suggests that some chimeric or humanized antibodies induce DAMP from tumor cells and may lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased ATP release

These experiments demonstrate that the humanized anti-CD 47mAb of the present disclosure induces increased release of Adenosine Triphosphate (ATP) from tumor cells as previously described using anthracycline chemotherapeutic drugs (Martins et al, 2014Cell Death and differentiation 21: 79-91).

Determination of the ATP Release from tumor cells by quantitative bioluminescence assay (molecular probes, Cat. A22066) as described by the manufacturerAnd (4) placing. Human Raji lymphoma cells (ATCC, Masnasas, Virginia; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 were used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for ATP release, cells were treated with 1 μ M of the chemotherapeutic anthracycline mitoxantrone. Cells were incubated at 37 ℃ for 24 hours, after which cell-free supernatants were collected and stored at-80 ℃. After all samples were collected, 10 μ l of each sample was tested by the ATP assay kit as described above. The final concentration was determined by comparing the experimental values to a standard curve and is shown as the concentration of ATP released (μ M) in response to antibody treated tumor cells. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPadPrism 6.

The humanized antibody increases the release of ATP from the tumor cells. As shown in figure 23, the amount of ATP released was significantly increased in all anti-CD 47mAb treated cultures compared to isotype control (p < 0.05). This increase in ATP release indicates that some chimeric or humanized antibodies induce ATP release from tumor cells and can lead to dendritic cell migration through their cognate purinergic receptors.

Humanized anti-CD 47mAb causes HMGB1 Release

These experiments demonstrate that the humanized anti-CD 47 mAbs of the present disclosure increase the release of non-histone chromatin protein high mobility group protein 1(HMGB1) from tumor cells as previously described using chemotherapeutic agents such as oxaliplatin (Tesnere et al, 2010Oncogene [ Oncogene ],29:482-491) and mitoxantrone (Michaud et al, 2011Science [ Science ]334: 1573-1577).

The release of HMGB1 protein from tumor cells was determined by an enzyme immunoassay (IBL International; Hamburg, Germany, Cat. ST 51011.) as described by the manufacturer, using either human Raji lymphoma cells (ATCC, Manassas, Virginia; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 the cells will be at less than 1X 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Purified humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) and control chimeric antibodies, were then transiently transfected from CHO cells as described above, and added at a final concentration of 10 μ g/ml. As a positive control for HMGB1 release, lange cells were treated with 1 μ M of the chemotherapeutic anthracycline mitoxantrone. Cells were incubated at 37 ℃ for 24 hours, after which cell-free supernatants were collected and stored at-80 ℃. After all samples were collected, 10 μ l of each sample was tested by HMGB1 ELISA as described above. The final concentration was determined by comparing the experimental values to a standard curve and reported as the concentration of HMGB1 released (ng/ml) in response to antibody-treated tumor cells. Results are presented as mean ± SEM and statistical significance was analyzed using ANOVA in GraphPad Prism 6.

As shown in figure 24, the humanized antibody increased the release of HMGB1 protein from tumor cells. The amount of HMGB1 protein released in all anti-CD 47mAb treated cultures was significantly increased (p <0.05) compared to isotype control except VLX9hum _06IgG2(ns, not significant). This increase in HMGB1 release indicates that some chimeric or humanized antibodies induce the release of DAMP from tumor cells and can lead to dendritic cell activation.

Humanized anti-CD 47mAb causes CXCL10 release

These experiments demonstrate that the humanized anti-CD 47mAb of the present disclosure increases the production and release of the chemokine CXCL10 from human tumor cells as previously described using anthracycline chemotherapeutic drugs (sisigu et al, 2014nat. med. [ natural medicine ]20(11): 1301-.

Release of CXCL10 from tumor cells was determined by an enzyme immunoassay (addi bio, catalog No. DIP100) as described by the manufacturer. Human Raji lymphoma cells (ATCC, Manassas, Va.; Cat. CCL-86) or other cell types expressing sufficient levels of CD47 will be used. Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 5% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Humanized antibodies as disclosed herein (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) purified by transient transfection from CHO cells as described above, as well as control chimeric antibodies were added at a final concentration of 10 μ g/ml. As a positive control for CXCL10 release, lange cells were treated with 1 μ M of chemotherapeutic anthracycline mitoxantrone. Cells were incubated at 37 ℃ for 24 hours, after which cell-free supernatants were collected and stored at-80 ℃. After all samples were collected, 10 μ l of each sample was tested by CXCL10 ELISA as described above. The final concentration was determined by comparing experimental values to a standard curve and is shown as the concentration of CXCL10 released (pg/ml) in response to antibody-treated tumor cells.

Some chimeric or humanized antibodies induce human tumor cells to release CXCL 10. As shown in figure 25, the amount of CXCL10 released was significantly increased in all anti-CD 47mAb treated cultures compared to isotype control (p < 0.05). This increase in CXCL10 release indicates that some chimeric or humanized antibodies induce CXCL10 release from tumor cells and indicate a role in recruiting immune cells to tumors.

Example 11

These studies were performed as described in example 10, except that a human Jurkat T ALL cell line (ATCC, Manassas, Virginia; catalog No. TIB-152) was used.

Humanized anti-CD 47mAb causes loss of mitochondrial membrane potential

As shown in figure 26, the humanized mabs (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) caused a significant increase in the percentage of cells with mitochondrial membrane depolarization (p <0.05) compared to isotype controls. This increase in the amount of mitochondrial membrane depolarization indicates that some chimeric or humanized antibodies induce cell death in human tumor cells.

Humanized anti-CD 47mAb causes increased cell surface calreticulin expression

As shown in figure 27, the humanized antibodies (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) induced pre-apoptotic exposure of calreticulin on the surface of tumor cells. The percentage of calreticulin-positive cells in all anti-CD 47mAb treated cultures was significantly increased (p <0.05) compared to isotype controls except VLX9hum _03 IgG2 (ns). This increase in calreticulin exposure on the cell surface suggests that some humanized antibodies induce DAMP from tumor cells and may lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased cell surface PDIA3 expression

As shown in figure 28, the percentage of PDIA3 positive cells in soluble anti-CD 47mAb (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) treated cultures was significantly increased (p <0.05) compared to the background obtained with the negative control, humanized isotype matched antibody. This increase in PDIA3 exposure on the cell surface suggests that some chimeric or humanized antibodies induce DAMP from tumor cells and may lead to phagocytosis of tumor cells and treatment of tumor antigens by innate immune cells.

Humanized anti-CD 47mAb causes increased cell surface HSP70 expression

As shown in figure 29, the percentage of HSP70 positive cells in anti-CD 47mAb (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) treated cultures was significantly increased (p <0.05) compared to those observed in isotype control treated cultures. Although each anti-CD 47mAb caused a statistically significant increase in HSP70 expression, mitoxantrone did not. This increase in HSP70 exposure on the cell surface suggests that some chimeric or humanized antibodies induce DAMP from tumor cells and may lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased cell surface HSP90 expression

As shown in figure 30, the percentage of HSP90 positive cells in soluble anti-CD 47mAb (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) treated cultures was significantly increased (p <0.05) compared to the background obtained with the negative control, humanized isotype matched antibody. This increase in HSP90 exposure on the cell surface suggests that some chimeric or humanized antibodies induce DAMP from tumor cells and may lead to phagocytosis of tumor cells by innate immune cells and treatment of tumor antigens.

Humanized anti-CD 47mAb causes increased ATP release

As shown in figure 31, the amount of ATP released was significantly increased (p <0.05) in the humanized anti-CD 47mAb (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) treated cultures compared to isotype controls. Although each anti-CD 47mAb caused a statistically significant increase in HSP70 expression, mitoxantrone did not (ns). This increase in ATP release will indicate that some chimeric or humanized antibodies induce ATP release from tumor cells and can lead to dendritic cell migration through their cognate purinergic receptors.

Humanized anti-CD 47mAb causes increased HMGB1 release

As shown in figure 32, the amount of HMGB1 protein released in anti-CD 47mAb (VLX4hum _01IgG4PE, VLX4hum _07IgG4PE, VLX8hum _11IgG4PE, VLX9hum _06IgG2, VLX9hum _08 IgG2, and VLX9hum _03 IgG2) treated cultures was significantly increased (p <0.05) compared to isotype controls other than VLX4hum _01IgG4PE (ns). This increase in HMGB1 release indicates that some chimeric or humanized antibodies induce DAMP from tumor cells and can lead to dendritic cell activation.

Example 12

Use of humanized anti-CD 47 Combination treatment of mAb and chemotherapy results in additive or synergistic effects

These experiments demonstrate that the humanized anti-CD 47 mabs of the present disclosure elicit additive or synergistic activity when combined with clinically relevant chemotherapeutic agents to induce immunogenic cell death in human tumor cells.

The additive activity/synergy of the combination drug was determined by combining increasing concentrations of humanized anti-CD 47 mAbVLX4hum _07IgG4PE and doxorubicin (sigma, PHR 1789). Human Jurkat cells (ATCC, Mass., Van.; Cat. TIB-152) or other cell types expressing sufficient levels of CD47 were used. Mixing the cells at a ratio of less than 1 × 10⁶Individual cells/mL were grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520),100 units/mL penicillin, 100. mu.g streptomycin (Sigma; Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

Jurkat cells were incubated with 0.03-10 μ g/ml VLX4hum _07IgG4PE alone, 0.3-100nM doxorubicin alone, or 0.03-10 μ g/ml VLX4hum _07IgG4PE and 0.3-100nM doxorubicin in a combined dose-responsive matrix in RPMI medium for 24 hours at 37 deg.C, after which the cells were harvested and assayed for phosphatidylserine on the cell surface using annexin V, 7-AAD, ER stress marker calreticulin. The supernatant was harvested for analysis of ATP release (as described above). Results are presented as mean ± SEM.

As shown in figure 33, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 34, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells. As shown in figure 35, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of calreticulin-positive cells. As shown in figure 36, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on ATP release.

Example 13

Hemagglutination of human blood red blood cells (hRBC)

A number of CD47 antibodies (including B6H12, BRIC126, MABL1, MABL2, CC2C6, 5F9) have been shown to cause blood coagulation (HA) of washed RBCs in vitro or in vivo (Petrova P. et al Cancer Res [ Cancer research ] 2015; 75(15 supplementations): abstract nr 4271; U.S. Pat. No. 9,045,541; Uno et al Oncol Rep. [ oncology report ]17:1189-94,2007; Kikuchi et al Biochem Biophys Res. Commun. [ biochemical and biophysics research communication ]315:912-8,2004; Sikic B. et al J Clin Oncol [ journal of clinical Oncology ] 2016; 34 (supplementations; abstract 3019)). Hemagglutination of hRBCs was assessed after incubation of hRBCs with different concentrations of chimeric and humanized VLX4, VLX8, and VLX9mAb in vitro, approximately as described in Kikuchi et al Biochem Biophys Res. Commun [ Biochemical and biophysical research Command ] (2004)315: 912-. Blood was obtained from healthy donors, diluted (1:50) in PBS/1mM EDTA/BSA and washed 3 times with PBS/EDTA/BSA. hRBC were added to a U-bottomed 96-well plate with equal volume of antibody (75. mu.l each) and incubated at 37 ℃ for 3 hours and at 4 ℃ overnight. Compact RBC precipitation was observed with antibodies that did not cause hemagglutination, and diffuse, hazy precipitation was observed with antibodies that caused hemagglutination.

As shown in figure 37A and tables 1 and 2, VLX4hum _01 IgG1 caused visible hemagglutination of hrbcs, whereas humanized VLX4hum _01IgG4PE mAb did not (mAb concentration 50 μ g/ml to 0.3 ng/ml). VLX4hum — 01IgG4PE confers a detectable deficiency in hemagglutination with additional desirable antibody properties and potential therapeutic benefits in cancer treatment.

As shown in fig. 37B and tables 1 and 2, chimeric antibody VLX8IgG4PE (xi) and humanized antibodies VLX8hum _08IgG4PE, VLX8hum _09 IgG4PE, and VLX8hum _10 IgG4PE caused visible hemagglutination of hrbcs, whereas VLX8 humanized abs VLX8hum _01IgG4PE, VLX8hum _02IgG4PE, VLX8hum _03 IgG4PE, and VLX8hum _11IgG4PE did not cause such hemagglutination (mAb concentration 50 μ g/ml to 0.3 ng/ml).

The humanized antibodies VLX4hum _01IgG4PE, VLX8hum _01IgG4PE, VLX8hum _02IgG4PE, VLX8hum _03 IgG4PE, and VLX8hum _11IgG4PE confer additional desirable antibody properties and potential therapeutic benefits in cancer therapy to a detectable deficiency in hemagglutination.

As shown in fig. 38A and 38B, the chimeric antibody VLX9 IgG2xi caused visible hemagglutination of hrbcs, whereas all humanized VLX9 mabs (except VLX9hum _07 IgG2) did not cause detectable hemagglutination (at concentrations from 50ug/ml to 0.3 pg/ml). However, the amount of detectable hemagglutination caused by VLX9hum _07 was reduced compared to VLX9 IgG2 chimeric mAb. Moreover, the VLX9 humanized mAb confers a reduction or lack of detectable hemagglutination to the additionally desired antibody properties and potential therapeutic benefits in cancer treatment.

Example 14

In vivo antitumor Activity

The purpose of this experiment was to demonstrate that VLX4, VLX8, and VLX9 humanized antibodies (e.g., VLX4_07 IgG4PE, VLX8_10 IgG4PE, and VLX9hum _08 IgG2) reduce the tumor burden in vivo in a lymphoma mouse xenograft model.

Rakiman burkitt lymphoma cells (ATCC # CCL-86, Magnesas, Virginia) were maintained in RPMI-1640 (Longsha (Lonza); Wolvaville, McFall) supplemented with 10% bovine fetal serum (FBS; Omega Scientific; Tarzana, Calif.) under an atmosphere of 5% CO 2. Cultures were expanded in tissue culture flasks.

5-6 week old female NSG (NOD-Cg-Prkdc) was obtained from Jackson laboratory (Barr Harbor, ME, Maine)^scidI12rg^tm1Wjl/SzJ). Mice were acclimated prior to treatment and housed in miniature isolation cages (laboratory Products, tin ford, terra) under specific pyrogen-free conditions. Mice were fed Teklad Global

2920x irradiated laboratory animal diet (Envigo, Formerly Harlan, Ind. Indianapolis, Ind.) and optionally autoclaved water. All procedures were performed under Institutional Animal Care and Use guidelines.

Using a catalyst containing 5X 10⁶0.1mL of 30% RPMI/70% Matrigel in Raji tumor cell suspension^TM(BD Biosciences; Bedford, MA, Mass.) mixtures were subcutaneously inoculated with the flanks of female NSG mice. Five days after inoculation, the width and length diameters of the tumors were measured using digital calipers. Tumor volume was calculated using the formula: tumor volume (mm)³)＝(a×b²/2) where "b" is the minimum diameter and "a" is the maximum diameter. Will have a diameter of 31-74mm³Can touch the tumor bodyThe pooled mice were randomly divided into 8-10/group and VLX9hum _08 or PBS (control) dosing was started at this time. Mice were treated by intraperitoneal injection with 5mg/kg antibody 5X/week for 4 weeks. Tumor volume and body weight were recorded twice weekly.

As shown in figure 39, treatment with humanized VLX4hum — 07IgG4PE significantly reduced tumor growth of the rasgi tumor (p <0.05, bilateral ANOVA), confirming anti-tumor efficacy in vivo.

As shown in figure 40, treatment with humanized anti-CD 47mAb VLX8hum _10 IgG4PE significantly reduced (p <0.0001, bilateral ANOVA) tumor growth of the lagigre tumor, confirming anti-tumor efficacy in vivo.

As shown in figure 41, treatment with humanized anti-CD 47mAb VLX9hum _08 IgG2 significantly reduced (p <0.05, bilateral ANOVA) tumor growth of the lagigold tumor, confirming anti-tumor efficacy in vivo.

Example 15

Effect on circulating Red blood cell parameters

The purpose of this experiment was to demonstrate that VLX9 humanized antibodies (table 2) (e.g., hum1017_08 IgG2) that do not bind human RBC in vitro do not cause a reduction in hemoglobin (Hg) or circulating RBC after administration to cynomolgus monkeys.

Female Chinese cynomolgus monkeys (Charles river Laboratories, Houston, Tex.) were used at 2.5-3 kg according to institute animal care and instructions. VLX9hum _08 IgG2 or vehicle (PBS) was administered as a1 hour intravenous infusion at a dose of 5mg/kg on day 1 and 15mg/kg on day 18 (3 animals/group). Hematological parameters were measured and compared/normalized to the mean values of control animals at day-7, day-3 (not shown), pre-dose, day 3, day 8, day 12, day 18 (pre-dose), day 20, day 25, day 29, day 35, and day 41 throughout the study. RBC and Hg values were lower in the VLX9hum _08 IgG2 group before treatment at day 0 than in the control group. There was minimal (< 10%) change in Hg (fig. 42A) or RBC count (fig. 42B) following treatment with either dose of VLX9hum _08 IgG2 compared to the control group, demonstrating that VLX9hum _08 IgG2 caused minimal reduction in RBC hematological parameters when administered to cynomolgus monkeys.

Example 16

Antibodies to CD47 modulate nitric oxide signaling

TSP1, which binds CD47, activates heterotrimeric G protein Gi, resulting in the inhibition of intracellular cyclic amp (camp) levels. In addition, the TSP1-CD47 pathway antagonizes the beneficial effects of the Nitric Oxide (NO) pathway in all vascular cells. The NO pathway consists of any of three enzymes (NOs I, NOs II, and NOs III) that produce the biologically active gas NO using arginine as a substrate. NO may act within the cell in which it is produced, or in neighboring cells, thereby activating the enzyme that produces the messenger molecule cyclic gmp (cgmp) (soluble guanosine cyclase). Proper function of the NO-cGMP pathway is important for protecting the cardiovascular system against stresses including, but not limited to, stresses due to trauma, inflammation, hypertension, metabolic syndrome, ischemia, and Ischemia Reperfusion Injury (IRI). In these cellular stress situations, inhibition of the NO/cGMP pathway by the TSP1/CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles. There are many situations in which ischemia and reperfusion injury cause or contribute to the poor outcome of disease, trauma, and surgery.

The purpose of these experiments was to demonstrate that the humanized anti-CD 47 mabs of the present disclosure exhibited the ability to reverse TSP 1-mediated inhibition of NO-stimulated cGMP synthesis, e.g., as previously described using a mouse monoclonal antibody against CD47, as disclosed by Isenberg et al (2006) j.biol.chem. [ journal of biochemistry ]281:26069-80, or alternatively other downstream markers of or effects guided by NO signaling, such as smooth muscle cell relaxation or platelet aggregation, as previously described by Miller et al (2010) Br j.pharmacol. [ journal of british pharmacology ] 154159: 1542-.

The method used to measure cGMP is described by the manufacturer (CatchPoint Cyclic-GMP fluorescence assay kit, Molecular Devices, Inc., Sunnyvale, Calif.). Jurkat JE6.1 cells (ATCC, Manassa, Virginia) will be usedS; directory number TIB-152) or other cell types that retain the NO-cGMP signaling pathway when grown in culture and exhibit a robust and reproducible inhibitory response to the TSP1 junction of CD 47. Cells were grown at a density of less than 1X 106 cells/mL in Iscove 'S modified Dulbecco' S medium containing 5% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma; Cat. No. P4222). For the cGMP assay, cells will be at 1X 10⁵The density of individual cells/mL of Iskoff 'S modified Du' S medium containing 5% (v/v) heat-inactivated bovine fetal serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222) was seeded in 96-well tissue culture plates for 24 hours and then transferred to serum-free medium overnight.

Then, a final concentration of 20ng/ml of humanized antibody purified from transient transfection of CHO cells as in example 3 above, as well as a control chimeric antibody, will be added, after 15 minutes, with 0 or 1 μ g/ml of human TSP1 (Athens Research and Technology, Asens, Ga., Asens, Cat. No. 16-20-201319). After an additional 15 minutes, the NO donor Diethylamine (DEA) nitric oxide (saemann Chemical, Ann Arbor, MI, michigan, catalog No. 82100) will be added to half of the wells at a final concentration of 1 μ M. After five minutes, the cells will be lysed with the buffer provided in the cGMP kit and an aliquot of each well will be assayed for cGMP content.

It is expected that some chimeric or humanized antibodies will reverse TSP1 inhibition of cGMP. The reversal will be complete (> 80%) or moderate (> 20% -80%). This cGMP-reversing TSP1 inhibition would confirm their ability to increase NO signaling and suggest their utility in protecting the cardiovascular system from stresses including, but not limited to, those due to trauma, inflammation, hypertension, metabolic syndrome, ischemia, and Ischemia Reperfusion Injury (IRI). Additional assay systems (e.g., smooth muscle cell contraction) are also expected to show that some chimeric or humanized antibody clones reverse the inhibitory effect of TSP on downstream effects caused by activation of NO signaling.

Example 17

Induction of cell death and DAMP expression by soluble CD47 antibody

The induction of cell death by soluble anti-CD 47mAb was measured in vitro (Manna et al J.Immunol. [ J.Immunol. ]. J.Immunol. ]]170:3544-3553, 2003; cancer Research by Manna et al],64:1026-1036,2004). For in vitro cell death assays, 1X 10 cells were incubated at 37 deg.C⁵Each transformed human ovarian cell (OV90 cell, ATCC, Mass.; Cat. CRL-11732) was incubated with the soluble humanized CD47mAb VLX4hum _07IgG4PE (0.03-3. mu.g/ml), VLX9hum _06IgG2 CD47 (1-100. mu.g/ml), and VLX8hum _11IgG4PE (0.03-3. mu.g/ml) for 24 hours. As cell death occurs, the mitochondrial membrane potential decreases, the inner lobes of the cell membrane invert, exposing Phosphatidylserine (PS) and calreticulin on the cell surface, and Propidium Iodide (PI) or 7-amino actinomycin D (7-AAD) can bind to nuclear DNA. To detect these cellular changes, cells were then stained with fluorescently labeled annexin V and PI or 7-amino-actinomycin D (7-AAD) (BD biosciences), rabbit monoclonal antibodies against calreticulin were conjugated with Alexa Fluor 647 (Ebos; Cat. No. ab196159), and signals were detected using an Attune flow cytometer (Life technologies). The increase in PS exposure was determined by measuring the percentage increase in annexin V signal and the percentage of dead cells was determined by measuring the percentage increase in PI or 7-AAD signal. Annexin V positivity was observed at the early stage of cell death (annexin V)⁺) Or annexin V positive/7-AAD negative (annexin V)⁺/7-AAD^-) Cells, and annexin V positive/7-AAD positive (annexin V)⁺/7-AAD⁺) The cell is a dead cell. By measuring the absence of PI or 7-AAD (calreticulin)⁺/7-AAD^-) The percentage increase in calreticulin-positive cells of (a) determines Calreticulin (CRT) exposure. Importantly for therapeutic purposes, these mabs directly induce cell death of tumor cells and do not require supplementation or intervention with other cells (e.g., NK cells, T cells, or macrophages) for killing. Thus, the mechanism is independent of other cells and Fc effector functions. Thus, therapeutic antibodies developed from these mabs can be engineered to reduce Fc effector functions such as ADCC and CDC and thus limit the possibility of side effects common to humanized mabs with intact Fc effector function.

As shown in FIGS. 43-45, soluble VLX4hum _07IgG4PE humanized mAb induced increased PS exposure and cell death of OV90 cells, as by being annexin V⁺/7-AAD^-(FIG. 43) and annexin V⁺/7-AAD⁺(FIG. 44) measured as% increase in cells. The percentage of cells in anti-CD 47 antibody treated cultures that were CRT +/7-AAD- (FIG. 45) was significantly increased (p) compared to isotype control<0.05 or greater).

As shown in figures 46-48, the soluble VLX9hum _06IgG2 humanized mAb induced increased PS exposure and cell death of OV90 cells as measured by the% increase in cells that were annexin V +/7-AAD- (figure 46) and annexin V +/7-AAD + (figure 47). The percentage of cells in anti-CD 47 antibody treated cultures that were CRT +/7-AAD- (figure 48) was significantly increased (p <0.05 or greater) compared to isotype control.

As shown in figures 49-51, soluble VLX8hum _11IgG4PE humanized mAb induced increased PS exposure and cell death of OV90 cells as measured by the% increase in cells for annexin V +/7-AAD- (figure 49) and annexin V +/7-AAD + (figure 50). The percentage of cells in anti-CD 47 antibody treated cultures that were CRT +/7-AAD- (figure 51) was significantly increased (p <0.05 or greater) compared to isotype control.

Induction of cell death, expression of DAMPs, and promotion of phagocytosis of susceptible cancer cells confer additional desirable antibody properties and therapeutic benefits in cancer therapy. This increase in calreticulin exposure on the cell surface indicates that VLX4hum _07IgG4PE, VLX9hum _06IgG2, and VLX8hum _11IgG4PE humanized CD47mAb induces DAMP from tumor cells, indicating further utility in stimulating tumor cell phagocytosis by innate immune cells and treatment of tumor antigens.

Example 18

Combination therapy of humanized anti-CD 47mAb (VLX4hum _07 IgG4PE) and chemotherapy resulted in additive or synergistic effects

The combined drug additive/synergistic effect was determined by combining increasing concentrations of humanized anti-CD 47mAb VLX4hum _07IgG4PE with doxorubicin (sigma, PHR1789), epirubicin (sigma, E9406), docetaxel (sigma, 01885), gemcitabine (sigma, 1288463), irinotecan (sigma, I1406), oxaliplatin (sigma, PHR 1528). Human OV10/315 cells (Gao and Lindberg, Journal of Biological Chemistry],1996). Mixing the cells at a ratio of less than 1 × 10⁶The density of individual cells/mL was grown in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest, Inc.; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma, Cat. No. P4222). For this assay, cells were plated at 1X 10⁵cells/mL RPMI-1640 medium at a density were plated in 96-well tissue culture plates containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222).

OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07IgG4PE alone, 0.05-0.42 μ M doxorubicin alone, or a combination dose response matrix of 0.03-1 μ g/ml VLX4hum _07IgG4PE and 0.05-0.42 μ M doxorubicin in RPMI medium for 24 hours at 37 deg.C, after which the cells were harvested and analyzed for phosphatidylserine using annexin V and DNA exposure by 7-AAD. Results are presented as mean ± SEM.

As shown in figure 52, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 53, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells.

OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07IgG4PE alone, 0.05-0.42 μ M epirubicin alone, or a combination dose response matrix of 0.03-1 μ g/ml VLX4hum _07IgG4PE and 0.05-0.42 μ M epirubicin in RPMI medium for 24 hours at 37 ℃ after which the cells were harvested and analyzed for phosphatidylserine using annexin V and DNA exposure by 7-AAD. Results are presented as mean ± SEM.

As shown in FIG. 54, some combinations of VLX4hum _07IgG4PE and epirubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 55, some combinations of VLX4hum _07IgG4PE and epirubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells.

OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07IgG4PE alone, 0.002-0.135 μ M docetaxel alone, or a combination dose response matrix of 0.03-1 μ g/ml VLX4hum _07IgG4PE and 0.002-0.135 μ M docetaxel in RPMI medium for 24 hours at 37 deg.C, after which the cells were harvested and analyzed for phosphatidylserine using annexin V and DNA exposure by 7-AAD. Results are presented as mean ± SEM.

As shown in FIG. 56, some combinations of VLX4 hum-07 IgG4PE and docetaxel caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 57, some combinations of VLX4hum _07IgG4PE and docetaxel caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells.

OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07IgG4PE alone, 0.003-0.3 μ M gemcitabine alone, or a combination dose response matrix of 0.03-1 μ g/ml VLX4hum _07IgG4PE and 0.003-0.3 μ M gemcitabine in RPMI medium for 24 hours at 37 deg.C, after which the cells were harvested and assayed for phosphatidylserine on the cell surface using annexin V, 7-AAD, ER stress marker calreticulin. Results are presented as mean ± SEM.

As shown in FIG. 58, some combinations of VLX4hum _07IgG4PE and gemcitabine caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 59, some combinations of VLX4hum _07IgG4PE and gemcitabine caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells. As shown in figure 60, some combinations of VLX4hum _07IgG4PE and gemcitabine caused additive or synergistic effects on the percentage of calreticulin-positive cells.

OV10/315 cells were incubated with 0.03-1. mu.g/ml VLX4hum _07IgG4PE alone, 0.63-51nM irinotecan alone or a combination dose-responsive matrix of 0.03-1. mu.g/ml VLX4hum _07IgG4PE and 0.63-51nM irinotecan in RPMI medium for 24 hours at 37 ℃ after which the cells were harvested and assayed for phosphatidylserine on the cell surface using annexin V, 7-AAD, ER stress marker calreticulin. Results are presented as mean ± SEM.

As shown in figure 61, some combinations of VLX4hum _07IgG4PE and irinotecan caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 62, some combinations of VLX4hum _07IgG4PE and irinotecan caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells. As shown in figure 63, some combinations of VLX4hum _07IgG4PE and irinotecan caused additive or synergistic effects on the percentage of calreticulin-positive cells.

OV10/315 cells were incubated with 0.03-1 μ g/ml VLX4hum _07IgG4PE alone, 0.65-52.8 μ M oxaliplatin alone or a combination dose response matrix of VLX4hum _07IgG4PE and 0.65-52.8 μ M oxaliplatin in 0.03-1 μ g/ml and in RPMI medium for 24 hours at 37 ℃, after which the cells were harvested and analyzed for phosphatidylserine using annexin V and DNA exposure by 7-AAD. Results are presented as mean ± SEM.

As shown in figure 64, some combinations of VLX4hum — 07IgG4PE and oxaliplatin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 65, some combinations of VLX4hum _07IgG4PE and oxaliplatin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells.

Example 19

Combination treatment of humanized anti-CD 47mAb (VLX9hum _06 IgG2) and chemotherapy resulted in additive or synergistic effects

The additive activity/synergy of the combination drugs was determined by combining increasing concentrations of the humanized anti-CD 47mAb VLX9hum _06IgG2 and doxorubicin (sigma, PHR 1789). Human Jurkat T ALL cell line (ATCC, Mass. Sas, Virginia; Cat. No. TIB-152) was used. Cells were grown at a density of less than 1X 106 cells/mL in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma; Cat. No. P4222). For this assay, cells were seeded at a density of 1X 105 cells/mL RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222) in 96-well tissue culture plates.

Jurkat cells were incubated with 1-100 μ g/ml VLX9hum _06IgG2 alone, 0.005-0.42 μ M doxorubicin alone, or a combination dose response matrix of 1-100 μ g/ml VLX9hum _06IgG2 and 0.005-0.42 μ M doxorubicin in RPMI medium for 24 hours at 37 deg.C, after which the cells were harvested and assayed for phosphatidylserine on the cell surface using annexin V, 7-AAD, ER stress marker calreticulin. Results are presented as mean ± SEM.

As shown in figure 66, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 67, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells. As shown in figure 68, some combinations of VLX4hum _07IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of calreticulin-positive cells.

Example 20

Combination therapy with humanized anti-CD 47mAb (VLX8hum _11 IgG4PE) and chemotherapy resulted in additive or synergistic effects Should be taken

The additive activity/synergy of the combination drugs was determined by combining increasing concentrations of the humanized anti-CD 47mAb VLX8hum _11IgG4PE and doxorubicin (sigma, PHR 1789). Human Jurkat T ALL cell line (ATCC, Mass. Sas, Virginia; Cat. No. TIB-152) was used. Cells were grown at a density of less than 1X 106 cells/mL in RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g mL streptomycin (Sigma; Cat. No. P4222). For this assay, cells were seeded at a density of 1X 105 cells/mL RPMI-1640 medium containing 10% (v/v) heat-inactivated fetal bovine serum (BioWest; Cat. No. S01520), 100 units/mL penicillin, 100. mu.g/mL streptomycin (Sigma; # P4222) in 96-well tissue culture plates.

Jurkat cells were incubated with 0.03-3 μ g/ml VLX8hum _11IgG4PE alone, 0.005-0.42 μ M doxorubicin alone, or a combination dose response matrix of 0.03-3 μ g/ml VLX8hum _11IgG4PE and 0.005-0.42 μ M doxorubicin in RPMI medium for 24 hours at 37 ℃, after which the cells were harvested and analyzed for phosphatidylserine on the cell surface using annexin V, 7-AAD, ER stress-labeled calreticulin, and cell supernatants were analyzed for HMGB1 release. Results are presented as mean ± SEM.

As shown in FIG. 69, some combinations of VLX8hum _11IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD negative (annexin V +/7-AAD-) cells. As shown in figure 70, some combinations of VLX8hum _11IgG4PE and doxorubicin caused additive or synergistic effects on the percentage of annexin V positive/7-AAD positive (annexin V +7-AAD +) dead cells. As shown in FIG. 71, the percentage of calreticulin-positive cells (calreticulin +/7-AAD-) caused additive or synergistic effects of some combinations of VLX8hum _11IgG4PE and doxorubicin. As shown in FIG. 72, VLX8hum _1¹Some combinations of IgG4PE and doxorubicin caused additive or synergistic effects on HMGB1 release.

Example 21

pH-dependent and independent binding of humanized anti-CD 47mAb

Some soluble anti-CD 47 mabs have been shown to bind tumor cells with greater affinity at acidic pH than at physiological pH. This additional property is expected to have advantages over mabs that bind CD47 with similar affinity at both acidic and physiological pH due to the acidic nature of the tumor microenvironment (Tannock and Rotin, Cancer Res [ Cancer research ] 1989; Song et al Cancer Drug Discovery and Development [ Cancer Drug Discovery and Development ] 2006; Chen and Pagel, Advan radio [ advanced radiology ] 2015).

The binding of soluble anti-CD 47mAb to recombinant Fc-CD47 was measured in vitro by surface plasmon resonance on Biacore 2000. Anti-human IgG (GE medical life sciences) is an amine coupled to CM5 chips on

flow cells

1 and 2. Will be in PBS-EP⁺Middle diluted recombinant Fc-CD47 was captured onto

flow cells

1 and 2. Used in HBS-EP⁺Running buffer (pH)7.5, 7, 6.5 or 6) diluted 0 to 1000nM of the humanized mAb VLX4hum _01Fab, VLX8hum _11Fab or VLX9hum _08Fab with a contact time of 180 seconds and a dissociation time of 300 seconds. Kinetic analysis of binding curves was performed using a 1:1 binding model. The association rates, dissociation rates, and dissociation constants for VLX4hum _01Fab, VLX8hum _11Fab, and VLX9hum _08Fab are shown in table 7 and demonstrate that VLX9hum _08 has pH-dependent binding to CD47, whereas VLX4hum _01 and VLX8hum _11 do not. This pH dependence confers additional desirable antibody properties and therapeutic benefits in cancer treatment.

Table 7.Determination of VLX4 by surface plasmon resonance Fab、VLX8 Fab and VLX9 Fab humanized mAb and recombinant Fc- Binding of CD 47.

Example 22

In vivo anti-tumor Activity in human xenograft model

The purpose of this experiment was to demonstrate that humanized antibodies to VLX4, VLX8, and VLX9 (e.g., VLX8hum — 10 IgG4PE) reduce tumor burden in a triple negative breast cancer mouse xenograft model in vivo.

MDA-MB-231 triple negative breast cancer cells (catalog number HTB-26TM, Marina, Virginia) were maintained in RPMI-1640 (Longsha, Wolff, Mass.) supplemented with 10% bovine fetal serum (FBS; Euromaca technologies; san Francisco, Calif.) under an atmosphere of 5% CO 2. Cultures were expanded in tissue culture flasks.

5-6 week old female NSG (NOD-Cg-Prkdc) was obtained from Jackson laboratory (Barr Harbor, ME, Maine)^scidI12rg^tm1Wjl/SzJ). Mice were acclimatized prior to treatment and in a mini-isolation cage (laboratory Products, Seaford (DE) Telawa, Tex.) under specific pyrogen-free conditionsMiddle-jiao captive breeding. Mice were fed Teklad Global

Using a catalyst containing 2X 10⁷0.2mL of an MDA-MB-231 tumor cell suspension 70% RPMI/30% Matrigel^TM(BD biosciences; Bedford, Mass.) the mixture was inoculated in situ into the mammary fat pad of female NSG mice. At 19 days post inoculation, 50 will have 55-179mm by random equilibration³The mice with accessible tumor volumes were randomly divided into 5 groups of 10 mice each. Tumor volume was calculated using the formula: tumor volume (mm)³)＝(a×b²/2) where "b" is the minimum diameter and "a" is the maximum diameter. VLX9hum _08 or PBS (control) administration was started at this point. Mice were treated by intraperitoneal injection for 5 weeks with 15mg/kg antibody 5X/week. Tumor volume and body weight were recorded twice weekly.

As shown in figure 73, treatment with humanized VLX8hum _10 IgG4PE significantly reduced tumor growth of MDA-MB-231 tumors (p <0.05, bilateral ANOVA), confirming anti-tumor efficacy in vivo.

Sequence listing

<110> ARCH ONCOLOGY, Inc

<120> therapeutic CD47 antibody

<130>VLX0008-401-PC

<150>US 62/475,032

<151>2017-03-22

<150>US 15/871,802

<151>2018-01-15

<150>US 62/475,036

<151>2017-03-22

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115

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115

<210>32

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115

<210>33

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115

<210>34

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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115

<210>35

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Gln Val Gln Leu Gln Gln Phe Gly Ala Glu Leu Ala Lys Pro Gly Ala

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Met Arg Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

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115

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Val Thr Val Ser Ser

115

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Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

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Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

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Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

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Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

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115

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Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

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Lys Asp Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210>39

<211>117

<212>PRT

<213> Intelligent (Homo sapiens)

<400>39

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Ala Gln Lys Phe

5055 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210>40

<211>117

<212>PRT

<213> Intelligent (Homo sapiens)

<400>40

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 7075 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210>41

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>41

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>42

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>42

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>43

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>43

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Asp Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>44

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>44

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>45

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>45

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 2530

Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>46

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<400>46

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210>47

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<400>47

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile AlaThr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>48

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<400>48

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210>49

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<400>49

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Asp Asp Val Gly Ile Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210>50

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>50

Asp Val Phe Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210>51

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>51

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>52

<211>112

<212>PRT

<213> Intelligent (Homo sapiens)

<400>52

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Asn Ile Val Gln Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210>53

<211>330

<212>PRT

<213> Intelligent (Homo sapiens)

<400>53

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>54

<211>330

<212>PRT

<213> Intelligent (Homo sapiens)

<400>54

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 4045

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>55

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<400>55

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210>56

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<400>56

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210>57

<211>377

<212>PRT

<213> Intelligent (Homo sapiens)

<400>57

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro

100 105 110

Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg

115 120 125

Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys

130 135 140

Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro

145 150 155 160

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

165 170 175

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

180 185 190

Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr

195 200 205

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

210 215 220

Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His

225 230 235 240

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

245 250 255

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln

260 265 270

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met

275 280 285

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

290 295 300

Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn

305 310 315 320

Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu

325 330 335

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile

340 345 350

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

355 360 365

Lys Ser Leu Ser Leu Ser Pro Gly Lys

370 375

<210>58

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<400>58

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

<210>59

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<400>59

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305310 315 320

Leu Ser Leu Ser Leu Gly

325

<210>60

<211>327

<212>PRT

<213> Intelligent (Homo sapiens)

<400>60

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210>61

<211>107

<212>PRT

<213> Intelligent (Homo sapiens)

<400>61

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>62

<211>329

<212>PRT

<213> Brown rat (Rattus norvegicus)

<400>62

Ala Arg Thr Thr Ala Pro Ser Val Tyr Pro Leu Val Pro Gly Cys Ser

1 5 10 15

Gly Thr Ser Gly Ser Leu Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Lys Trp Asn Ser Gly Ala Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Gly Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Ser Ser Gln Thr Val

65 70 75 80

Thr Cys Ser Val Ala His Pro Ala Thr Lys Ser Asn Leu Ile Lys Arg

85 90 95

Ile Glu Pro Arg Arg Pro Lys Pro Arg Pro Pro Thr Asp Ile Cys Ser

100 105 110

Cys Asp Asp Asn LeuGly Arg Pro Ser Val Phe Ile Phe Pro Pro Lys

115 120 125

Pro Lys Asp Ile Leu Met Ile Thr Leu Thr Pro Lys Val Thr Cys Val

130 135 140

Val Val Asp Val Ser Glu Glu Glu Pro Asp Val Gln Phe Ser Trp Phe

145 150 155 160

Val Asp Asn Val Arg Val Phe Thr Ala Gln Thr Gln Pro His Glu Glu

165 170 175

Gln Leu Asn Gly Thr Phe Arg Val Val Ser Thr Leu His Ile Gln His

180 185 190

Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys

195 200 205

Asp Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Arg Gly Lys

210 215 220

Ala Arg Thr Pro Gln Val Tyr Thr Ile Pro Pro Pro Arg Glu Gln Met

225 230 235 240

Ser Lys Asn Lys Val Ser Leu Thr Cys Met Val Thr Ser Phe Tyr Pro

245 250 255

Ala Ser Ile Ser Val Glu Trp Glu Arg Asn Gly Glu Leu Glu Gln Asp

260 265 270

Tyr Lys Asn Thr Leu Pro ValLeu Asp Ser Asp Glu Ser Tyr Phe Leu

275 280 285

Tyr Ser Lys Leu Ser Val Asp Thr Asp Ser Trp Met Arg Gly Asp Ile

290 295 300

Tyr Thr Cys Ser Val Val His Glu Ala Leu His Asn His His Thr Gln

305 310 315 320

Lys Asn Leu Ser Arg Ser Pro Gly Lys

325

<210>63

<211>107

<212>PRT

<213> Brown rat (Rattus norvegicus)

<400>63

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Met Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Thr Val Val Cys Phe Val Asn Asn Phe

20 25 30

Tyr Pro Arg Asp Ile Ser Val Lys Trp Lys Ile Asp Gly Ser Glu Gln

35 40 45

Arg Asp Gly Val Leu Asp Ser Val Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Ser Leu Thr Lys Val Glu Tyr Glu

65 70 75 80

Arg His Asn Leu Tyr Thr Cys Glu Val Val His Lys Thr Ser Ser Ser

85 90 95

Pro Val Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210>64

<211>323

<212>PRT

<213> Rabbit (Oryctolagus cuniculus)

<400>64

Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly

1 5 10 15

Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn

35 40 45

Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys

65 70 75 80

Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala

85 90 95

Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly

100 105110

Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

115 120 125

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln

130 135 140

Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val

145 150 155 160

Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile

165 170 175

Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg Gly

180 185 190

Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile

195 200 205

Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val

210 215 220

Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser

225 230 235 240

Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu

245 250 255

Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala

260 265270

Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val

275 280 285

Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met

290 295 300

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser

305 310 315 320

Pro Gly Lys

<210>65

<211>104

<212>PRT

<213> Rabbit (Oryctolagus cuniculus)

<400>65

Arg Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp

1 5 10 15

Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr

20 25 30

Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr

35 40 45

Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr

50 55 60

Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser

65 70 75 80

His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val

85 90 95

Gln Ser Phe Asn Arg Gly Asp Cys

100

<210>66

<211>293

<212>PRT

<213> Rabbit (Oryctolagus cuniculus)

<400>66

Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly

1 5 10 15

Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe

20 25 30

Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala

35 40 45

Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp

50 55 60

Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp

65 70 75 80

Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala

85 90 95

Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr

100 105 110

Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu

115 120 125

Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu

130 135 140

Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe

145 150 155 160

Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr

165 170 175

Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val

180 185 190

Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr

195 200 205

Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His

210 215 220

Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala

225 230 235 240

Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu

245 250 255

Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile

260 265 270

Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr

275 280 285

Met Lys Phe Val Glu

290

<210>67

<211>219

<212>PRT

<213> Intelligent (Homo sapiens)

<400>67

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>68

<211>219

<212>PRT

<213> Intelligent (Homo sapiens)

<400>68

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Asn Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 2530

Asn Gly Asn Thr Tyr Leu Gly Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>69

<211>219

<212>PRT

<213> Intelligent (Homo sapiens)

<400>69

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Asp Asp Val Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>70

<211>214

<212>PRT

<213> Intelligent (Homo sapiens)

<400>70

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Asp Asp Val Gly Ile Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>71

<211>219

<212>PRT

<213> Intelligent (Homo sapiens)

<400>71

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Asn Ile Val Gln Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>72

<211>214

<212>PRT

<213> Intelligent (Homo sapiens)

<400>72

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala SerGln Ser Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>73

<211>219

<212>PRT

<213> Intelligent (Homo sapiens)

<400>73

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Arg Gln Ser Ile Val His Thr

20 25 30

Asn Gly Asn Thr Tyr Leu Gly Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>74

<211>219

<212>PRT

<213> Intelligent (Homo sapiens)

<400>74

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser SerThr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>75

<211>214

<212>PRT

<213> Intelligent (Homo sapiens)

<400>75

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>76

<211>214

<212>PRT

<213> little mouse (Mus musculus)

<400>76

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210>77

<211>219

<212>PRT

<213> little mouse (Mus musculus)

<400>77

Asp Val Phe Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Ile Val Gln Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Phe His Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>78

<211>444

<212>PRT

<213> little mouse (Mus musculus)

<400>78

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 1015

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Val Lys Arg Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Val Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>79

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>79

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Ala Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>80

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>80

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Ala Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>81

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>81

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val GluCys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>82

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>82

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Gln Val Thr IleSer Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>83

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>83

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>84

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>84

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Gln Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Leu Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 9095

Ala Arg Gly Gly Tyr Tyr Val Tyr Asp Tyr Trp Gly Gln Ala Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>85

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>86

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>86

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Arg Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435440

<210>87

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>87

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala LeuGly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>88

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>88

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Tyr Pro Tyr Asn Asp Gly Ile Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Tyr Tyr Val Pro Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>89

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>89

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Ser Pro Ser Phe

50 55 60

Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>90

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>90

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 1015

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>91

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>91

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>92

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>92

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>93

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>93

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>94

<211>444

<212>PRT

<213> Intelligent (Homo sapiens)

<400>94

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg ValThr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>95

<211>444

<212>PRT

<213> little mouse (Mus musculus)

<400>95

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Asn Gln Ser His Gly Lys Ser Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Arg Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

210 215 220

Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

245 250 255

Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe

260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

275 280 285

Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

290 295 300

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

305 310 315 320

Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala

325 330 335

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln

340 345 350

Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

355 360 365

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

385 390 395 400

Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu

405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

420 425 430

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210>96

<211>443

<212>PRT

<213> little mouse (Mus musculus)

<400>96

Gln Val Gln Leu Gln Gln Phe Gly Ala Glu Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Gln Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Thr Asp Pro Arg Thr Asp Tyr Thr Glu Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Ala Ala Asp Arg Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Arg Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

8590 95

Ala Gly Gly Gly Arg Val Gly Leu Gly Tyr Trp Gly His Gly Ser Ser

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>97

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>97

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>98

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>98

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>99

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>99

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu

1 5 10 15

Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>100

<211>443

<212>PRT

<213> Intelligent (Homo sapiens)

<400>100

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asp Pro Leu Asn Gly Asp Thr Thr Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Lys Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn

180 185 190

Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro

210 215 220

Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

225 230 235 240

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

245 250 255

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn

260 265 270

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

275 280 285

Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val

290 295 300

Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

305 310 315 320

Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys

325 330 335

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

340 345 350

Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

355 360 365

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

370 375 380

Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe

385 390 395 400

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

405 410 415

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

420 425 430

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440

<210>101

<211>326

<212>PRT

<213> Intelligent (Homo sapiens)

<400>101

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly

325

Claims

1. A method of treating cancer in a subject, the method comprising administering to the subject an anti-CD 47 antibody, or antigen-binding fragment thereof, and a second anti-cancer agent, the administration of the anti-CD 47 antibody, or antigen-binding fragment thereof, and the second anti-cancer agent resulting in an increase in Immunogenic Cell Death (ICD) of tumor cells in the subject as compared to the administration of the anti-CD 47 antibody alone.

2. The method of claim 1, wherein the Immunogenic Cell Death (ICD) feature comprises:

a. increased Adenosine Triphosphate (ATP) release; and

b. cell surface calreticulin expression on human tumor cells.

3. The method of claim 1, wherein the second anticancer agent is a chemotherapeutic agent.

4. The method of claim 3, wherein the chemotherapeutic agent may be selected from the group consisting of: anthracyclines, platins, taxol, topoisomerase inhibitors, antimetabolites, antitumor antibiotics, mitotic inhibitors, and alkylating agents.

5. The method of claim 4, wherein the anthracycline is selected from the group consisting of doxorubicin, epirubicin, daunorubicin, and idarubicin.

6. The method of claim 5, wherein the anthracycline is doxorubicin.

7. The method of claim 1, wherein the cancer is ovarian cancer.

8. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells; and

d. inducing death of human tumor cells;

wherein the monoclonal antibody or antigen binding fragment thereof has one or more of the following characteristics:

e. causing an increase in cell surface calreticulin expression on human tumor cells;

f. causing an increase in Adenosine Triphosphate (ATP) release from human tumor cells;

g. causing an increase in the release of high mobility group box 1 protein (HMGB1) from human tumor cells;

h. causing an increase in annexin a1 release from human tumor cells;

i. causing an increase in the release of type I interferon from human tumor cells;

j. causes an increase in C-X-C motif chemokine ligand 10(CXCL10) release from human tumor cells;

k. causing an increase in the expression of cell surface protein disulfide isomerase A3(PDIA3) on human tumor cells;

causing an increase in the expression of cell surface heat shock protein 70(HSP70) on human tumor cells; and

causing an increase in the expression of cell surface heat shock protein 90(HSP90) on human tumor cells.

9. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. inducing death of human tumor cells; and

e. does not cause detectable human red blood cell (hRBC) agglutination;

f. causing an increase in cell surface calreticulin expression on human tumor cells;

g. causing an increase in Adenosine Triphosphate (ATP) release from human tumor cells;

h. causing an increase in the release of high mobility group box 1 protein (HMGB1) from human tumor cells;

i. causing an increase in annexin a1 release from human tumor cells;

j. causing an increase in the release of type I interferon from human tumor cells;

k. causes an increase in C-X-C motif chemokine ligand 10(CXCL10) release from human tumor cells;

causing an increase in the expression of cell surface protein disulfide isomerase a3(PDIA3) on human tumor cells;

10. The monoclonal antibody or antigen-binding fragment thereof of claim 9, which is a chimeric antibody or a humanized antibody.

11. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 9-10, wherein the monoclonal antibody or antigen-binding fragment thereof has minimal binding to hRBCs.

12. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 9-11, wherein the monoclonal antibody or antigen-binding fragment thereof, wherein binding to normal human cells is reduced, including but not limited to endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubule epithelial cells, human peripheral blood CD3+ cells, and human peripheral blood mononuclear cells).

13. The monoclonal antibody, or antigen-binding fragment thereof, of claims 9-12, wherein the monoclonal antibody, or antigen-binding fragment thereof, has greater affinity for CD47 at acidic pH than at physiological pH.

14. The monoclonal antibody or antigen binding fragment thereof of any one of claims 9-13, comprising a heavy chain variable domain comprising variable heavy chain CDR1, variable heavy chain CDR2, and variable heavy chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 1, 2, 3;

wherein said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id nos: 4, 5, 6; and

wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, and SEQ ID NO 10.

15. The monoclonal antibody, or antigen binding fragment thereof, of claim 14, further comprising a light chain variable domain comprising variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 11, 12, 13, 14;

wherein said variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id nos: 15, 16, 17 and

wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id nos: 18, 19 and 20.

16. The monoclonal antibody or antigen-binding fragment thereof of claim 15, comprising a combination of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), and variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR3(LCDR3), wherein the combination is selected from the group consisting of:

(iv) HCDR1 comprising SEQ ID NO. 3, HCDR2 comprising SEQ ID NO. 6, HCDR3 comprising SEQ ID NO. 10, LCDR1 comprising SEQ ID NO. 14, LCDR2 comprising SEQ ID NO. 17, LCDR3 comprising SEQ ID NO. 20.

17. The monoclonal antibody or antigen binding fragment thereof of claim 16, comprising a heavy chain variable domain (V) having an amino acid sequence selected from the group consisting of_H) The group consisting of: amino acid sequences of: 21, 22, 23, 27, 33, 34, 36, 38, 39, and 40, and optionally comprises a light chain variable domain (V) having an amino acid sequence selected from the group consisting of_L) The group consisting of: SEQ ID NO 41, 42, 43, 44, 48, 49, 51 and 52.

18. The monoclonal antibody or antigen binding fragment thereof of claim 17, comprising a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) Wherein the combination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 44;

(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 33 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48;

(iv) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 34 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 49;

(v) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 36 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52;

(vi) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52; and

(vii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 39 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52.

19. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which:

a. in combination with the human CD47,

b. blocks the binding of SIRP α to human CD47,

c. increase the phagocytosis of human tumor cells,

d. inducing death of human tumor cells; and

e. causing a reduction in human red blood cell (hRBC) agglutination.

i. causing an increase in annexin a1 release from human tumor cells;

20. The monoclonal antibody or antigen-binding fragment thereof of claim 19, which is a chimeric antibody or a humanized antibody.

21. The monoclonal antibody, or antigen binding fragment thereof, of any one of claims 19-20, wherein the monoclonal antibody, or antigen binding fragment thereof, has reduced hRBC binding.

22. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 19-21, wherein the monoclonal antibody or antigen-binding fragment thereof, wherein binding to normal human cells is reduced, including but not limited to endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubule epithelial cells, human peripheral blood CD3+ cells, and human peripheral blood mononuclear cells).

23. The monoclonal antibody, or antigen-binding fragment thereof of any one of claims 19-22, wherein the monoclonal antibody, or antigen-binding fragment thereof, has greater affinity for human CD47 at acidic pH than at physiological pH.

24. The monoclonal antibody, or antigen binding fragment thereof, of any one of claims 19-23, comprising a heavy chain variable domain comprising variable heavy chain CDR1, variable heavy chain CDR2, and variable heavy chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 1 and 3 of SEQ ID NO,

wherein said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id nos: 4 and 6; and

wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 10.

25. The monoclonal antibody, or antigen binding fragment thereof, of claim 24, further comprising a light chain variable domain comprising variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 11 and 14;

wherein said variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id nos: 15 and 17; and

wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id nos: 18 and 20, respectively.

26. The monoclonal antibody or antigen binding fragment thereof of claim 25, comprising a combination of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), and variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR3(LCDR3), wherein the combination is selected from the group consisting of:

(i) HCDR1 comprising SEQ ID NO. 1, HCDR2 comprising SEQ ID NO. 4, HCDR3 comprising SEQ ID NO. 8, LCDR1 comprising SEQ ID NO. 11, LCDR2 comprising SEQ ID NO. 15, and LCDR3 comprising SEQ ID NO. 18;

(ii) HCDR1 comprising SEQ ID NO. 3, HCDR2 comprising SEQ ID NO. 6, HCDR3 comprising SEQ ID NO. 10, LCDR1 comprising SEQ ID NO. 14, LCDR2 comprising SEQ ID NO. 17, and LCDR3 comprising SEQ ID NO. 20.

27. The monoclonal antibody or antigen binding fragment thereof of claim 26, comprising a heavy chain variable domain (V) having an amino acid sequence selected from the group consisting of_H) The group consisting of: amino acid sequences of: 24 and 37, and optionally comprises a light chain variable domain (V) having an amino acid sequence selected from the group consisting of_L) The group consisting of: SEQ ID NO 43 and SEQ ID NO 52.

28. The monoclonal antibody or antigen binding fragment thereof of claim 27, comprising a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) Wherein the combination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 24 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 43; and

(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 37 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 52.

29. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which:

a. in combination with the human CD47,

b. blocks the binding of SIRP α to human CD47,

c. increasing phagocytosis of human tumor cells

d. Inducing death of human tumor cells; and

e. with reduced hRBC binding.

i. causing an increase in annexin a1 release from human tumor cells;

30. The monoclonal antibody or antigen-binding fragment thereof of claim 29, which is a chimeric antibody or a humanized antibody.

31. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 29-30, wherein the monoclonal antibody, or antigen-binding fragment thereof, wherein binding to normal human cells is reduced, including but not limited to endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripheral blood CD3+ cells, and human peripheral blood mononuclear cells).

32. The monoclonal antibody, or antigen-binding fragment thereof of any one of claims 29-31, wherein the monoclonal antibody, or antigen-binding fragment thereof, has greater affinity for human CD47 at acidic pH than at physiological pH.

33. The monoclonal antibody, or antigen binding fragment thereof, of any one of claims 29-32, comprising a heavy chain variable domain comprising variable heavy chain CDR1, variable heavy chain CDR2, and variable heavy chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 1 and 2 SEQ ID NO;

wherein said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID:4 and SEQ ID:5, and

wherein said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO 7, SEQ ID NO 8 and SEQ ID NO 9.

34. The monoclonal antibody, or antigen binding fragment thereof, of claim 33, further comprising a light chain variable domain comprising variable light chain CDR1, variable light chain CDR2, and variable light chain CDR3, wherein said variable chain CDR1 comprises an amino acid sequence selected from the group consisting of seq id nos: 11, 12 and 13;

wherein said variable light chain CDR2 comprises an amino acid sequence selected from the group consisting of seq id nos: 15 and 16; and

wherein said variable light chain CDR3 comprises an amino acid sequence selected from the group consisting of seq id nos: 18 and 19 SEQ ID NO.

35. The monoclonal antibody or antigen-binding fragment thereof of claim 34, comprising a combination of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), and variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR3(LCDR3), wherein the combination is selected from the group consisting of:

(i) HCDR1 comprising SEQ ID NO. 1, HCDR2 comprising SEQ ID NO. 4, HCDR3 comprising SEQ ID NO. 7, LCDR1 comprising SEQ ID NO. 11, LCDR2 comprising SEQ ID NO. 15, LCDR3 comprising SEQ ID NO. 18; and

(ii) HCDR1 comprising SEQ ID NO. 2, HCDR2 comprising SEQ ID NO. 5, HCDR3 comprising SEQ ID NO. 9, LCDR1 comprising SEQ ID NO. 13, LCDR2 comprising SEQ ID NO. 16, LCDR3 comprising SEQ ID NO. 19.

36. The monoclonal antibody or antigen binding fragment thereof of claim 35, comprising a heavy chain variable domain (V) having an amino acid sequence selected from the group consisting of_H) The group consisting of: amino acid sequences of: SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:33, and optionally comprises a light chain variable domain (V) having an amino acid sequence selected from the group consisting of_L) The group consisting of: SEQ ID NO 44 and SEQ ID NO 48.

37. The monoclonal antibody or antigen binding fragment thereof of claim 36, comprising a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) Wherein the combination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 44;

(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 44; and

(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 33 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 48.

38. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which:

a. in combination with the human CD47,

b. blocks the binding of SIRP α to human CD47,

c. increase the phagocytosis of human tumor cells,

d. does not cause detectable human red blood cell (hRBC) agglutination; and

e. with minimal hRBC binding.

i. causing an increase in annexin a1 release from human tumor cells;

39. The monoclonal antibody or antigen-binding fragment thereof of claim 38, which is a chimeric antibody or a humanized antibody.

40. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 38-39, wherein the monoclonal antibody, or antigen-binding fragment thereof, wherein binding to normal human cells is reduced, including but not limited to endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubular epithelial cells, human peripheral blood CD3+ cells, and human peripheral blood mononuclear cells).

41. The monoclonal antibody, or antigen-binding fragment thereof of any one of claims 38-40, wherein the monoclonal antibody, or antigen-binding fragment thereof, has greater affinity for human CD47 at acidic pH than at physiological pH.

42. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 38-41, comprising a combination of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), and variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR3(LCDR3), wherein the combination is:

(i) HCDR1 comprising SEQ ID NO. 3, HCDR2 comprising SEQ ID NO. 6, HCDR3 comprising SEQ ID NO. 10, LCDR1 comprising SEQ ID NO. 14, LCDR2 comprising SEQ ID NO. 17, LCDR3 comprising SEQ ID NO. 20.

43. The monoclonal antibody or antigen binding fragment thereof of claim 42, comprising a heavy chain variable domain (V) having an amino acid sequence selected from the group consisting of_H) The group consisting of: amino acid sequences of: 38, 39, 40 and optionally comprises a light chain variable domain (V) having an amino acid sequence selected from the group consisting of_L) The group consisting of: SEQ ID NO 51 and SEQ ID NO 52.

44. The monoclonal antibody or antigen binding fragment thereof of claim 43, comprising a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) Wherein the combination is selected from the group consisting of:

(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 38 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51;

(ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 39 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51; and

(iii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 40 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 52.

45. The monoclonal antibody or antigen-binding fragment thereof of claim 1, which:

a. binds to human CD 47;

b. block binding of SIRP α to human CD 47;

c. increase phagocytosis of human tumor cells;

d. does not cause detectable human red blood cell (hRBC) agglutination; and

e. with reduced hRBC binding.

i. causing an increase in annexin a1 release from human tumor cells;

46. The monoclonal antibody or antigen-binding fragment thereof of claim 45, which is a chimeric antibody or a humanized antibody.

47. The monoclonal antibody, or antigen-binding fragment thereof, of any one of claims 45-46, wherein the monoclonal antibody, or antigen-binding fragment thereof, wherein binding to normal human cells is reduced, including but not limited to endothelial cells, skeletal muscle cells, epithelial cells, and peripheral blood mononuclear cells (e.g., human aortic endothelial cells, human skeletal muscle cells, human microvascular endothelial cells, human renal tubule epithelial cells, human peripheral blood CD3+ cells, and human peripheral blood mononuclear cells).

48. The monoclonal antibody, or antigen-binding fragment thereof of any one of claims 45-47, wherein the monoclonal antibody, or antigen-binding fragment thereof, has greater affinity for human CD47 at acidic pH than at physiological pH.

49. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 45-48, comprising a combination of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), and variable heavy chain CDR3(HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2(LCDR2), and variable light chain CDR3(LCDR3), wherein the combination is:

50. The monoclonal antibody or antigen binding fragment thereof of claim 49, comprising a heavy chain variable domain (V)_H) And a light chain variable domain (V)_L) Wherein the combination is:

(i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 36 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO 51.

51. The monoclonal antibody, or antigen-binding fragment thereof, of claim 1 that binds CD47, wherein the antibody or antigen-binding fragment comprises a combination of variable heavy chain CDR1(HCDR1), variable heavy chain CDR2(HCDR2), and variable heavy chain CDR3(HCDR3), wherein the combination is selected from the group consisting of:

(i) HCDR1 comprising SEQ ID NO. 1, HCDR2 comprising SEQ ID NO. 4, HCDR3 comprising SEQ ID NO. 7;

(ii) HCDR1 comprising SEQ ID NO. 1, HCDR2 comprising SEQ ID NO. 4, HCDR3 comprising SEQ ID NO. 8;

(iii) HCDR1 comprising SEQ ID NO 2, HCDR2 comprising SEQ ID NO 5, HCDR3 comprising SEQ ID NO 9; and

(iv) HCDR1 comprising SEQ ID NO 3, HCDR2 comprising SEQ ID NO 6, HCDR3 comprising SEQ ID NO 10.

52. The monoclonal antibody or antigen binding fragment of claim 51, further comprising a light chain variable domain comprising a combination of variable light chain CDR3(LCDR3), variable light chain CDR2(LCDR2), and variable light chain CDR1(LCDR1) selected from the group consisting of:

(i) LCDR1 comprising SEQ ID NO. 11, LCDR2 comprising SEQ ID NO. 15, LCDR3 comprising SEQ ID NO. 18;

(ii) LCDR1 comprising SEQ ID NO. 12, LCDR2 comprising SEQ ID NO. 16, LCDR3 comprising SEQ ID NO. 19;

(iii) LCDR1 comprising SEQ ID NO. 13, LCDR2 comprising SEQ ID NO. 16, LCDR3 comprising SEQ ID NO. 19; and

(iv) LCDR1 comprising SEQ ID NO. 14, LCDR2 comprising SEQ ID NO. 17, LCDR3 comprising SEQ ID NO. 20.

53. The monoclonal antibody or antigen-binding fragment of claim 52, comprising a combination of variable heavy chain CDR sequences and a combination of variable light chain CDR sequences selected from the group consisting of:

54. The monoclonal antibody or antigen binding fragment of claim 53, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable domain (V) selected from the group consisting of_H) And a light chain variable domain (V)_L) A combination of (a), the group consisting of:

(x) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 26 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 44;

(xi) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 27 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 44;

(xxxiv) A heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 40 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 51;

wherein the V_HThe amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the V_LThe amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.

55. The monoclonal antibody of any one of claims 51-54, comprising at least one heavy chain and at least one light chain selected from the group consisting of:

(xxiii) A heavy chain comprising the amino acid sequence of SEQ ID NO 92 and a light chain comprising the amino acid sequence of SEQ ID NO 72;

56. A method of preventing or treating cancer in a subject, the method comprising:

administering to the subject a combination of an anti-CD 47 antibody, or antigen-binding fragment thereof, and a second anti-cancer agent, the administration of the combination resulting in an increased therapeutic effect as compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.

57. The method of claim 56, wherein the therapeutic effect increases tumor cell death as compared to monotherapy administration of the anti-CD 47 antibody or a second anticancer agent.

58. The method of claim 56, wherein the therapeutic effect increases cell surface calreticulin expression of human tumor cells as compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.

59. The method of claim 56, wherein the therapeutic effect increases ATP release from human tumor cells as compared to monotherapy administration of the anti-CD 47 antibody or the second anticancer agent.

60. The method of claim 56, wherein the second anticancer agent is a chemotherapeutic agent.

61. The method of claim 60, wherein the chemotherapeutic agent may be selected from the group consisting of: anthracyclines, platins, taxol, topoisomerase inhibitors, antimetabolites, antitumor antibiotics, mitotic inhibitors, and alkylating agents.

62. The method of claim 61, wherein the anthracycline is selected from the group consisting of doxorubicin, epirubicin, daunorubicin, and idarubicin.

63. The method of claim 61, wherein the platinum is selected from the group consisting of oxaliplatin, cisplatin, and carboplatin.

64. The method of claim 61, wherein the taxol is selected from the group consisting of paclitaxel and docetaxel.

65. The method of claim 61, wherein the topoisomerase inhibitor is selected from the group consisting of irinotecan, topotecan, etoposide, and mitoxantrone.

66. The method of claim 61, wherein the antimetabolite is selected from 5-FU, capecitabine, cytarabine, gemcitabine, and pemetrexed.

67. The method of claim 61, wherein the mitotic inhibitor is vinorelbine, vinblastine, and vincristine.

68. The method of claim 61, wherein the alkylating agent is selected from the group consisting of temozolomide.

69. The method of claim 56, wherein the anti-cancer agent is bortezomib or carfilzomib.

70. The method of claim 56, wherein the cancer is ovarian cancer.

71. The method of claim 1, wherein said cancer is selected from the group consisting of: leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular cancer, gallbladder cancer, cholangiocarcinoma, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, endocrine adenocarcinoma, adrenal gland cancer, pituitary gland cancer, skin cancer, soft tissue cancer, blood vessel cancer, brain cancer, nerve cancer, eye cancer, meninges cancer, oropharyngeal cancer, hypopharynx cancer, cervical cancer, and uterine cancer, glioblastoma, medulloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and sarcoma.

72. The method of claim 67, wherein said leukemia is selected from the group consisting of: systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T-cell-ALL, Acute Myelogenous Leukemia (AML), myelogenous leukemia, Chronic Lymphocytic Leukemia (CLL), Multiple Myeloma (MM), Chronic Myelogenous Leukemia (CML), myeloproliferative disorders/neoplasms, myelodysplastic syndromes, monocytic leukemia, and plasma cell leukemia; wherein said lymphoma is selected from the group consisting of: histiocytic and T-cell lymphomas, B-cell lymphomas, including hodgkin's and non-hodgkin's lymphomas such as low grade/follicular non-hodgkin's lymphoma (NHL), cellular lymphoma (FCC), Mantle Cell Lymphoma (MCL), Diffuse Large Cell Lymphoma (DLCL), Small Lymphocytic (SL) NHL, intermediate grade/follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, large mass NHL, and waldenstrom's macroglobulinemia; and wherein said sarcoma is selected from the group consisting of: osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

73. A method of increasing Immunogenic Cell Death (ICD) of cancer cells in a cancer patient by administering an anti-CD 47 antibody or antigen binding fragment thereof, and a second anti-cancer agent to the cancer patient, the method resulting in increased Immunogenic Cell Death (ICD) of the cancer cells as compared to the administration of the anti-CD 47 antibody alone.

74. A method of increasing Immunogenic Cell Death (ICD) of cancer cells in a cancer patient by administering to the cancer patient an anti-CD 47 antibody or antigen binding fragment thereof, and a second anti-cancer agent, the method resulting in an increase in therapeutic effect as compared to monotherapy administration of the anti-CD 47 antibody or the second anti-cancer agent.