CN111201031A - anti-CD 47x anti-mesothelin antibodies and methods of use thereof - Google Patents

Abstract

The present invention also relates to novel bispecific antibodies with different specificity for each binding site of an immunoglobulin molecule, wherein one of the binding sites is specific for CD47 and the second binding site is specific for Mesothelin (MSLN).

Description

anti-CD 47x anti-mesothelin antibodies and methods of use thereof

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application No. 62/511,669 filed on 26.5.2017 and U.S. provisional application No. 62/550,387 filed on 25.8.2017. The contents of each of these applications are incorporated herein by reference in their entirety.

Sequence listing incorporated by reference

The contents of a file named "NOVI-044 _001US _ SequenceListing _ ST 25" created at 22.5/2018 and having a size of 268KB are incorporated herein by reference in their entirety.

Background

CD47 or integrin-associated protein (IAP) is a ubiquitous 50kDa transmembrane Glycoprotein with Multiple Functions in Cell-Cell communication, which interacts with various ligands such as integrin, SIRP α (signal-regulating protein α), SIRP γ and thrombospondin (Oldenborg, P.A., CD47: A Cell Surface protein Multiple Functions of hematoidic in blood Cells and diseases, ISRN blood 2013; 2013:614619; Soto-Pantoja DR, et al, therapeutic interactions for targeting the said intermediate Cell Surface 2012 CD47 (operator CD47), Expert Optin targets 2013 Jan; 17: 1) biological 89-167, et al, human inlet 597. 3. inlet of therapeutic tissue J. the expression of human tissue J.7. interaction of human tissue J.7. the invention also discloses a new protein.

The widespread expression of CD47 in healthy tissue raises the issue of therapeutic safety and efficacy in that, firstly, targeting CD47 with neutralizing monoclonal Antibodies (Mab) can affect healthy tissue, leading to severe toxicity as shown in preclinical studies with mice and cynomolgus monkeys (Willingham SB, et al, Proc Natl Acad Sci U S a. 2012 Apr 24;109(17):6662-7; weiskorf K, et al, Engineered SIRP α Variants as immunethereutical addjuvants to Antibodies, science 2013 Jul 5;341(6141):88-91), and secondly, even by using alternative forms, can avoid or mitigate the mediated severe toxicity (weiskpf K, et al, science.2013 (Jul 5; 341: 6141) CD 88-91), the widespread expression of CD47 leading to rapid elimination of the target-mediated drug-mediated potency via rapid pharmacokinetic distribution, 47-mediated drug binding, and decreased potency.

Thus, there is a need for antibodies and therapies that can target CD47 and overcome these difficulties.

Summary of The Invention

The present invention also provides bispecific antibodies that recognize CD47 and mesothelin CD47 (Cluster of differentiation 47) functions as a "do't eat me" signal for phagocytes and is known to be overexpressed (immune evasion) by many tumors, CD47 interacts with SIRP α (which is overexpressed on phagocytes), CD47 down-regulates phagocytic activity, CD47 inhibits Dendritic Cell (DC) maturation and activation, CD47 also participates in processes such as, for example, apoptosis, survival, proliferation, adhesion, migration, and regulation of angiogenesis, blood pressure, tissue perfusion, and/or platelet homeostasis.

CD47 is also implicated in cancer. For example, CD47 is overexpressed in a variety of hematologic and solid malignancies. CD47 is a proven cancer stem cell/tumor initiating cell marker. It is believed that CD47 overexpression may help tumor cells escape immune surveillance and be killed by innate immune cells. High levels of CD47 are also associated with poor clinical outcomes in cancers such as, for example, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, and/or glioma. Thus, targeting CD47 would be useful in treating, delaying progression of, or otherwise ameliorating the symptoms of cancer.

Mesothelin (MSLN) is expressed at relatively low levels in normal tissues. Mesothelin is highly expressed in several types of solid tumors, such as malignant mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, and endometrial carcinoma, biliary tract gastric cancer, and prostate cancer, as compared to normal tissue. Tumor mesothelin expression has often been associated with increased tumor invasiveness and poor clinical prognosis. Thus, targeting mesothelin would be useful in treating, delaying progression of, or otherwise ameliorating the symptoms of cancer.

The present invention also provides bispecific antibodies comprising at least a first arm specific for CD47, in some embodiments the first arm is specific for at least human CD47 in some embodiments the first arm recognizes human CD47 and is also cross-reactive with at least one other non-human CD47 protein (such as, but not limited to, non-human primate CD47, e.g., cynomolgus monkey CD47 and/or rodent CD 47.) in some embodiments these anti-CD 47 monoclonal antibodies inhibit the interaction between CD47 and signal-regulatory protein α (SIRP α) in some embodiments these bispecific antibodies inhibit the interaction between human CD47 and human SIRP α the invention also includes antibodies that bind to the same epitope as the specific antibodies disclosed herein and inhibit the interaction between CD47 and SIRP α, e.g., between human CD47 and human SIRP α.

The invention also provides bispecific antibodies that recognize CD47 and a second target. The present invention allows the identification, generation and purification of bispecific antibodies that are indistinguishable in sequence from standard antibodies and in which one of the binding sites is specific for CD47 and the second binding site is specific for another target, such as a Tumor Associated Antigen (TAA). In some embodiments, the TAA is an antigen expressed on the cell surface of a cancer cell. In some embodiments, the cancer cell is selected from a lung cancer cell, a bronchial cancer cell, a prostate cancer cell, a breast cancer cell, a colorectal cancer cell, a pancreatic cancer cell, an ovarian cancer, a leukemia cancer cell, a lymphoma cancer cell, an esophageal cancer cell, a liver cancer cell, a urinary and/or bladder cancer cell, a kidney cancer cell, an oral cancer cell, a pharyngeal cancer cell, a uterine cancer cell, and/or a melanoma cancer cell.

Bispecific antibodies of the invention that bind at least CD47 and fragments thereof are useful for modulating, blocking, inhibiting, reducing, antagonizing, neutralizing, or otherwise interfering with the functional activity of CD47 functional activity of CD47 includes, but is not limited to, interacting with SIRP α when the level of CD47-SIRP α interaction in the presence of the antibody is reduced by at least 95%, e.g., 96%, 97%, 98%, 99% or 100%, as compared to the level of CD47-SIRP α interaction in the absence of binding to the antibody described herein, the antibody is considered to fully modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with CD47-SIRP α interaction.

In some embodiments, a bispecific antibody exhibits "balanced" affinity for each of two targets. In other embodiments, the bispecific antibody exhibits "unbalanced" affinity for each of the two targets. For example, in an anti-CD 47/MSLN bispecific antibody, the affinity of the anti-MSLN arm is increased. For example, in an anti-CD 47/MSLN bispecific antibody, the affinity of the anti-CD 47 arm is reduced. For example, in an anti-CD 47/MSLN bispecific antibody, the affinity of the anti-MSLN arm is increased and the affinity of the anti-CD 47 arm is decreased. These unbalanced affinity bispecific antibodies are useful, for example, to improve selectivity for a target cell or group of target cells.

In some embodiments, the affinity of the anti-MSLN arm increases by at least 100-fold after affinity maturation. In some embodiments, the affinity of the anti-CD 47 arm is reduced by at least 2-fold after affinity de-maturation (dissociation). For example, in some embodiments, the anti-CD 47 arm exhibits an affinity for CD47 that is about 2-fold to less than 100-fold after affinity de-maturation.

In some embodiments, the first arm amino acid sequence comprises the variable heavy chain complementarity determining region 1(CDRH1) amino acid sequence of SEQ ID NO:225, the variable heavy chain complementarity determining region 2(CDRH2) amino acid sequence of SEQ ID NO:226, the variable heavy chain complementarity determining region 3(CDRH3) amino acid sequence of SEQ ID NO:227, the variable light chain complementarity determining region 1(CDRL1) amino acid sequence selected from SEQ ID NO: 228-.

In some embodiments, the first arm amino acid sequence comprises the variable heavy chain amino acid sequence of SEQ ID NO 114 and the variable light chain amino acid sequence selected from SEQ ID NOs 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, and 206.

In some embodiments, the first arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:240, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:242, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 254.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 56.

In some embodiments, the second arm amino acid sequence comprises the heavy chain amino acid sequence of SEQ ID NO. 2 and the light chain amino acid sequence selected from the group consisting of SEQ ID NO. 98, 100, 102, 104, 106, 108, and 110.

In some embodiments, the second arm amino acid sequence comprises the variable heavy chain amino acid sequence of SEQ ID NO 114 and the variable light chain amino acid sequence selected from SEQ ID NO 212, 214, 216, 218, 220, 222, and 224.

In some embodiments, the second arm amino acid sequence comprises the variable heavy chain complementarity determining region 1(CDRH1) amino acid sequence of SEQ ID NO:225, the variable heavy chain complementarity determining region 2(CDRH2) amino acid sequence of SEQ ID NO:226, the variable heavy chain complementarity determining region 3(CDRH3) amino acid sequence of SEQ ID NO:227, the variable light chain complementarity determining region 1(CDRL1) amino acid sequence selected from SEQ ID NO:282-287, the variable light chain complementarity determining region 2(CDRL2) amino acid sequence selected from SEQ ID NO:288-293, and the variable CDRL complementarity determining region 3(CDRL3) amino acid sequence selected from SEQ ID NO: 294-300.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:282, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:288, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 294.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:283, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:289, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 295.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:284, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:290, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 296.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:285, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:291, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 297.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:286, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:292, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 298.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:287, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:293, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 299.

In some embodiments, the second arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:282, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:288, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 300.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising an amino acid sequence selected from SEQ ID NO 212, 214, 216, 218, 220, 222, and 224.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 212.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:114 and a variable light chain comprising the amino acid sequence of SEQ ID NO: 214.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 216.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 218.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 220.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 222.

In some embodiments, the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 224.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising an amino acid sequence selected from SEQ ID NO. 98, 100, 102, 104, 106, 108, and 110.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 98.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 100.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 102.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 104.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 106.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 108.

In some embodiments, the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 110.

In some embodiments, the first arm amino acid sequence comprises the variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, the variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, the variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, the variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:240, the variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:242, and the variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO:254 and the second arm amino acid sequence comprises the variable light chain complementarity determining region 1(CDRL1) amino acid sequence selected from SEQ ID NO:282-287, the variable light chain complementarity determining region 2(CDRL2) amino acid sequence selected from SEQ ID NO:288-293, and a variable light chain complementarity determining region 3(CDRL3) amino acid sequence selected from the group consisting of SEQ ID NO: 294-300.

In some embodiments, the first arm amino acid sequence comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:240, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:242, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO:254 and the second arm amino acid sequence comprises variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:282, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:288, and variable light chain complementarity determining region 294 comprising SEQ ID NO:294 The variable light chain complementarity determining region 3(CDRL3) of the sequence.

In some embodiments, the first arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and the second arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:283, CDRL2 comprising the amino acid sequence of SEQ ID NO:289, and l3 comprising the amino acid sequence of SEQ ID NO: 295.

In some embodiments, the first arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and the second arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:284, CDRL2 comprising the amino acid sequence of SEQ ID NO:290, and l3 comprising the amino acid sequence of SEQ ID NO: 225.

In some embodiments, the first arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and the second arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:285, CDRL2 comprising the amino acid sequence of SEQ ID NO:291, and l3 comprising the amino acid sequence of SEQ ID NO: 297.

In some embodiments, the first arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and the second arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:286, CDRL2 comprising the amino acid sequence of SEQ ID NO:292, and l3 comprising the amino acid sequence of SEQ ID NO: 298.

In some embodiments, the first arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and the second arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:287, CDRL2 comprising the amino acid sequence of SEQ ID NO:293, and l3 comprising the amino acid sequence of SEQ ID NO: 299.

In some embodiments, the first arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and the second arm amino acid sequence comprises CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, CDRL1 comprising the amino acid sequence of SEQ ID NO:282, CDRL2 comprising the amino acid sequence of SEQ ID NO:288, and l3 comprising the amino acid sequence of SEQ ID NO: 300.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID No. 114 and a variable light chain comprising the amino acid sequence of SEQ ID No. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID No. 114 and a variable light chain comprising an amino acid sequence selected from SEQ ID NOs 212, 214, 216, 218, 220, 222, and 224.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 212.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 214.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 216.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 218.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 220.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 222.

In some embodiments, the first arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO. 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO. 224.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising an amino acid sequence selected from SEQ ID NO. 98, 100, 102, 104, 106, 108, and 110.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 98.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 100.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 102.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 104.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 106.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 108.

In some embodiments, the first arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 168, and the second arm amino acid sequence comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 110.

In some embodiments, the bispecific antibody comprises two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.

In some embodiments, at least a portion of the first light chain is of the kappa type and at least a portion of the second light chain is of the lambda type. In some embodiments, the first light chain comprises at least one kappa constant region. In some embodiments, the first light chain further comprises a kappa variable region. In some embodiments, the first light chain further comprises a λ variable region. In some embodiments, the second light chain comprises at least one lambda constant region. In some embodiments, the second light chain further comprises a λ variable region. In some embodiments, the second light chain further comprises a kappa variable region. In some embodiments, the first light chain comprises a kappa constant region and a kappa variable region, and wherein the second light chain comprises a lambda constant region and a lambda variable region.

In some embodiments, the constant and variable framework region sequences are human.

Bispecific antibodies of the invention are produced using any method known in the art, such as, but not limited to, using cross-linked fragments, cell hybridomas, and/or any of a variety of recombinant forms, such as, but not limited to, ligated antibody fragments, forced heterodimers (forced heterodimers), and or single domain-based recombinant forms examples of bispecific forms include, but are not limited to, bispecific IgG based on Fab arm exchange (Gramer et al, 2013 MAbs.5(6)), CrossMab forms (Klein C et al, 2012MAbs 4(6)), various forms based on forced heterodimerization methods such as SEED technology (Davis JH et al, 2010Protein Eng Des Sel.23 (195) 202), electrostatic manipulation (Gunasekakaran K et al, J Biol chem. 2015 (85) (Im37: 46) or incorporated into a hole (knob-into-Pro-11, No-Pro-11, No-Pro-35, No-Pro-11, No-Pro-35, No-Pro-9, No-Pro-9, No-9, No. 23, No. 9.

In some embodiments, the bispecific antibody has a specificity that is different at each binding site and comprises two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different. In some embodiments, at least a portion of the first light chain is of the kappa type and at least a portion of the second light chain is of the lambda type. In some embodiments, the first light chain comprises at least one kappa constant region. In some embodiments, the first light chain further comprises a kappa variable region. In some embodiments, the first light chain further comprises a λ variable region. In some embodiments, the second light chain comprises at least one lambda constant region. In some embodiments, the second light chain further comprises a λ variable region. In some embodiments, the second light chain further comprises a kappa variable region. In some embodiments, the first light chain comprises a kappa constant region and a kappa variable region, and the second light chain comprises a lambda constant region and a lambda variable region. In some embodiments, the constant and variable framework region sequences are human.

The present invention provides monoclonal antibodies that bind MSLN. These antibodies are collectively referred to herein as anti-MSLN monoclonal antibodies or anti-MSLN mabs. Preferably, the monoclonal antibody is specific for at least human MSLN. In some embodiments, a monoclonal antibody that recognizes human MSLN is also cross-reactive with at least one other non-human MSLN protein (such as, by way of non-limiting example, a non-human primate MSLN, e.g., cynomolgus monkey MSLN, and/or a rodent MSLN).

In some embodiments, the anti-MSLN monoclonal antibody comprises the variable heavy chain complementarity determining region 1(CDRH1) amino acid sequence of SEQ ID NO:225, the variable heavy chain complementarity determining region 2(CDRH2) amino acid sequence of SEQ ID NO:226, the variable heavy chain complementarity determining region 3(CDRH3) amino acid sequence of SEQ ID NO:227, the variable light chain complementarity determining region 1(CDRL1) amino acid sequence selected from SEQ ID NO:282-287, the variable light chain complementarity determining region 2(CDRL2) amino acid sequence selected from SEQ ID NO:288-293, and the variable light chain complementarity determining region 3(CDRL3) amino acid sequence selected from SEQ ID NO: 294-300.

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:282, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:288, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 294.

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:283, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:289, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 295.

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO:225 (CDRH1), variable heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO:226 (CDRH2), variable heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO:227 (CDRH3), variable light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO:284 (CDRL1), variable light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO:290 (CDRL2), and variable light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO:296 (CDRL 3).

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:285, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:291, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 297.

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO:225 (CDRH1), variable heavy chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO:226 (CDRH2), variable heavy chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO:227 (CDRH3), variable light chain complementarity determining region 1 comprising the amino acid sequence of SEQ ID NO:286 (CDRL1), variable light chain complementarity determining region 2 comprising the amino acid sequence of SEQ ID NO:292 (CDRL2), and variable light chain complementarity determining region 3 comprising the amino acid sequence of SEQ ID NO:298 (CDRL 3).

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:287, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:293, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 299.

In some embodiments, the anti-MSLN monoclonal antibody comprises variable heavy chain complementarity determining region 1(CDRH1) comprising the amino acid sequence of SEQ ID NO:225, variable heavy chain complementarity determining region 2(CDRH2) comprising the amino acid sequence of SEQ ID NO:226, variable heavy chain complementarity determining region 3(CDRH3) comprising the amino acid sequence of SEQ ID NO:227, variable light chain complementarity determining region 1(CDRL1) comprising the amino acid sequence of SEQ ID NO:282, variable light chain complementarity determining region 2(CDRL2) comprising the amino acid sequence of SEQ ID NO:288, and variable light chain complementarity determining region 3(CDRL3) comprising the amino acid sequence of SEQ ID NO: 300.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID No. 114 and a variable light chain comprising an amino acid sequence selected from SEQ ID NOs 212, 214, 216, 218, 220, 222, and 224.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 212.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 214.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 216.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 218.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 220.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 222.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 224.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 2 and a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 98, 100, 102, 104, 106, 108, and 110.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 98.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 100.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 102.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 104.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 106.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 108.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 110.

In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the anti-MSLN monoclonal antibody comprises a variable light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the variable light chain portion of an amino acid sequence selected from SEQ ID NOs 98, 100, 102, 104, 106, 108, and 110. In some embodiments, the anti-MSLN monoclonal antibody comprises a variable heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID No. 114, and a variable light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a variable light chain amino acid sequence selected from SEQ ID NOs 212, 214, 216, 218, 220, 222, and 224.

In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID No. 2. In some embodiments, the anti-MSLN monoclonal antibody comprises a light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from SEQ ID NOs 98, 100, 102, 104, 106, 108, and 110. In some embodiments, the anti-MSLN monoclonal antibody comprises a heavy chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the amino acid sequence of SEQ ID No. 114, and a light chain amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to an amino acid sequence selected from SEQ ID nos. 98, 100, 102, 104, 106, 108, and 110.

In some embodiments, the anti-MSLN monoclonal antibody comprises the heavy chain amino acid sequence of SEQ ID NO 2 and a light chain amino acid sequence selected from the group consisting of SEQ ID NO 98, 100, 102, 104, 106, 108, and 110.

The invention also provides monovalent antibodies that bind MSLN. These antibodies are collectively referred to herein as anti-MSLN monovalent antibodies or anti-MSLN monovalent (monov) mabs. The monovalent antibodies of the invention comprise one arm that specifically recognizes MSLN and a second arm referred to herein as a dummy arm (dummy arm). The prosthetic arm includes an amino acid sequence that does not bind to or otherwise cross-react with a human protein. In some embodiments, the prosthetic arm includes an amino acid sequence that does not bind to or otherwise cross-react with human proteins found in whole blood. In some embodiments, the prosthetic arm comprises an amino acid sequence that does not bind to or otherwise cross-react with a human protein found in the solid tissue. Preferably, the monovalent antibody is specific for at least human MSLN. In some embodiments, a monovalent antibody that recognizes human MSLN is also cross-reactive to at least one other non-human MSLN protein (such as, but not limited to, a non-human primate MSLN, e.g., cynomolgus monkey MSLN and/or rodent MSLN).

The invention also provides bispecific antibodies that recognize MSLN and a second target.

The bispecific antibodies of the invention that recognize MSLN and a second target are produced using any method known in the art, such as, but not limited to, the use of cross-linked fragments, cell hybridomas, and/or any of a variety of recombinant forms, such as, but not limited to, linked antibody fragments, forced heterodimers (forced heterodimers), and or single domain-based recombinant forms. The present invention allows the identification, generation and purification of bispecific antibodies that are indistinguishable in sequence from standard antibodies and in which one of the binding sites is specific for MSLN and the second binding site is specific for another target, such as a Tumor Associated Antigen (TAA). The unmodified nature of the antibodies of the invention allows them to have advantageous preparation and biochemical characteristics similar to standard monoclonal antibodies.

In some embodiments, the bispecific antibody has a specificity that is different at each binding site and comprises two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.

In some embodiments, at least a portion of the first light chain is of the kappa type and at least a portion of the second light chain is of the lambda type. In some embodiments, the first light chain comprises at least one kappa constant region. In some embodiments, the first light chain further comprises a kappa variable region. In some embodiments, the first light chain further comprises a λ variable region. In some embodiments, the second light chain comprises at least one lambda constant region. In some embodiments, the second light chain further comprises a λ variable region. In some embodiments, the second light chain further comprises a kappa variable region. In some embodiments, the first light chain comprises a kappa constant region and a kappa variable region, and the second light chain comprises a lambda constant region and a lambda variable region. In some embodiments, the constant and variable framework region sequences are human.

For example, the monoclonal, monovalent and/or bispecific antibodies of the invention can be used in methods of treating, preventing and/or delaying the progression of, or slowing symptoms associated with, aberrant CD47 and/or aberrant CD47-SIRP α expression and/or activity by administering the antibodies of the invention to a subject in need of such treatment or prevention.

In some embodiments, the monoclonal, monovalent and/or bispecific antibodies of the invention are useful in methods of treating, preventing and/or delaying progression of, or alleviating symptoms of, cancer or other neoplastic disease by administering an antibody of the invention to a subject in which treatment or prevention is desired. For example, the monoclonal, monovalent and/or bispecific antibodies described herein can be used to treat hematological malignancies and/or solid tumors. For example, monoclonal, monovalent, and/or bispecific antibodies described herein can be used to treat CD47⁺Tumor mesothelin⁺Tumors and combinations thereof. By way of non-limiting example, the monoclonal, monovalent and/or bispecific antibodies described herein may be used to treat non-hodgkin's lymphoma (NHL), Acute Lymphocytic Leukemia (ALL), Acute Myelogenous Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), chronic myelogenous leukemia (CLL), and the likeDisease (CML), Multiple Myeloma (MM), breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer (including pancreatic adenocarcinoma), lung cancer (including lung adenocarcinoma), leiomyoma, leiomyosarcoma, kidney cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary tract gastric cancer, and prostate cancer. Solid tumors include, for example, breast, ovarian, lung, pancreatic, prostate, melanoma, colorectal, lung, head and neck, bladder, esophageal, liver, and kidney tumors.

In some embodiments, the monoclonal, monovalent and/or bispecific antibodies described herein are used in combination with one or more additional agents or combinations of additional agents. Suitable additional agents include pharmaceutical and/or surgical therapies currently used for the intended applications such as, for example, cancer, inflammation, and/or autoimmune diseases. In some embodiments, a monoclonal, monovalent and/or bispecific antibody may be used in combination with rituximab.

In some embodiments, the monoclonal, monovalent and/or bispecific antibody and the additional agent are formulated into a monotherapy composition and the monoclonal, monovalent and/or bispecific antibody and the additional agent are administered simultaneously. Alternatively, the monoclonal, monovalent and/or bispecific antibody and the additional agent are separated from each other, e.g., each formulated in a separate therapeutic composition, and the monoclonal, monovalent and/or bispecific antibody and the additional agent are administered simultaneously, or the monoclonal, monovalent and/or bispecific antibody and the additional agent are administered at different times during a treatment regimen. For example, the monoclonal, monovalent and/or bispecific antibody is administered prior to administration of the additional agent, the monoclonal, monovalent and/or bispecific antibody is administered after administration of the additional agent, or the monoclonal, monovalent and/or bispecific antibody and the additional agent are administered in an alternating manner. As described herein, the monoclonal, monovalent and/or bispecific antibody and the additional agent are administered in a single dose or in multiple doses.

Pathologies treated and/or prevented using the antibodies of the invention include, for example, cancer or any other disease or disorder associated with aberrant CD47 expression and/or activity.

The pharmaceutical composition according to the present invention may comprise the antibody of the present invention and a carrier. These pharmaceutical compositions may be included in a kit, such as a diagnostic kit, for example.

Brief Description of Drawings

FIGS. 1A-1G are a series of graphs depicting binding experiments of the CD47/MSLN κ λ body to the corresponding MSLN mAb. In fig. 1A-1G, binding of biAb O25 (fig. 1A), biAb O30 (fig. 1B), biAb O32 (fig. 1C), biAb O35 (fig. 1D), biAb O37 (fig. 1E), biAb O38 (fig. 1F) and biAb O41 (fig. 1G) to human MSLN-transfected CHO cells (CHO-huMSLN, left), cynomolgus monkey MSLN-transfected CHO cells (CHO-cyMSLN, mid) and untransfected CHO cells (right) was assessed by flow cytometry in dose-response and expressed as mean fluorescence intensity. Comparison between the CHO-humSLN and CHO-cyMSLN binding profiles shows that the CD47/MSLN kappa lambda bodies and MSLN mAbs of the invention cross-react with cynomolgus monkey MSLN and that they bind with comparable affinity to MSLN from both species.

FIGS. 2A-2F are a series of graphs depicting antibody-dependent cellular phagocytosis (ADCP) induced by the CD47/MSLN κ λ body, compared to a reference mAb targeting CD47 or mesothelin. In fig. 2A-2F, the levels of induced ADCP are shown as the percentage of phagocytosis induced by increasing concentrations of bispecific antibody (biAbs), CD47 mAb B6H12 (with a human IgG1 moiety), MSLN mAb amateximab (amatuximab), and the corresponding anti-MSLN monovalent antibody (i.e., antibody with anti-MSLN antibody arm and non-binding antibody arm), as assessed by flow cytometry. Phagocytosis was performed with human macrophages differentiated from peripheral blood mononuclear cells and two target cell lines NCI-N87 (fig. 2A-2C) and HPAC (fig. 2E and 2F). The cell surface expression levels of CD47 and mesothelin of NCI-N87 cells were 43,000 and 27,000, respectively. The cell surface expression levels of CD47 and mesothelin for HPAC cells were 105,000 and 13,000, respectively.

Figures 3A-3J are a series of graphs depicting ADCP activity induced by CD47/MSLN κ λ bodies compared to matched anti-MSLN mabs and monovalent antibodies. Phagocytosis of human macrophages by two target cell lines NCI-N87 (FIGS. 3A-3E) and Caov-3 (FIGS. 3F-3J) was imaged and quantified using the CellInsight CX5 high-content screening platform. Figures 3A-3J depict phagocytic indices, which correspond to the average number of target cells ingested by 100 macrophages. The cell surface expression levels of CD47 and mesothelin of NCI-N87 cells were 43,000 and 27,000, respectively. The cell surface expression levels of CD47 and mesothelin for Caov-3 cells were 220,000 and 38,000, respectively.

Figures 4A-4C are a series of graphs depicting dose-responsive ADCC experiments with the CD47/MSLN κ λ body compared to the baseline MSLN monoclonal antibody amazemab. ADCC assays were performed using whole human PBMC as effector cells and three Cr 51-loaded MSLN-positive target cell lines NCI-N87 (FIG. 4A), NCI-H226 (FIG. 4B), and HepG2-MSLN (FIG. 4C). Target cell killing was assessed using Cr 51-releasing cell-based assays. The percentage of ADCC was determined as specific Cr51 release, calculated using the formula: % ADCC = ((sample cpm-nonspecific lysis control cpm)/(total lysis control cpm-negative control cpm)) x 100%. The CD47/MSLN κ λ bodies induced dose-dependent killing of target cells, which was significantly higher than with the baseline MSLN mAb.

FIGS. 5A and 5B show the anti-tumor activity of 5 CD47/MSLN κ λ antibodies against the corresponding CD47 monovalent antibody and the benchmark monoclonal antibody, CD47 Mab B6H12 (on a human IgG1 background), and MSLN mAb amateximab. In FIGS. 5A and 5B, HepG2-MSLN cells were implanted subcutaneously in NOD/SCID mice. Antibody treatment was started after 15 days. (FIG. 5A) tumor growth progression. Tumor growth was measured three times a week and shown as mean tumor volume +/-SEM (n =7) for each group. Statistical significance was determined at endpoint (D55) using one-way ANOVA followed by multiple comparison test (Tukey's multiple comparison), p-value: p <0.05, p < 0.01; ns, not significant. (FIG. 5B) Tumor Growth Inhibition (TGI). The percentage of TGI compared to isotype control was determined based on tumor volume at the endpoint using the following formula: % TGI = {1- [ (Tt-T0)/(Vt-V0) ] } x 100; wherein Tt = median tumor volume treated at time t; t0 = median tumor volume treated at time 0; vt = median tumor volume of the control at time t, and V0 = median tumor volume of the control at time 0. biAb treatment significantly reduced tumor growth compared to controls. Furthermore, 4 of the 5 biabs tested demonstrated more efficacy than the MSLN reference mAb amateximab.

FIGS. 6A and 6B show the anti-tumor activity of biabO38 against two MSLN expressing ovarian cancer cell lines. In FIGS. 6A and 6B, tumor OVCAR3 and CaOV3 cells were implanted subcutaneously in NOD/SCID mice, respectively. Antibody treatment was started after 1 day. Figure 6A shows OVCAR3 tumor growth progression. Fig. 6B shows the CaOV3 tumor growth process. Tumor growth was measured three times per week and shown as mean tumor volume +/-SEM (n =6 or 7) for each group. biAbO38 treatment prevented tumor growth (compared to control IgG).

Detailed description of the invention

CD47 or integrin-associated Protein (IAP) are ubiquitous 50kDa transmembrane glycoproteins with multiple functions in Cell-Cell communication, which interact with multiple ligands such as integrins and/or SIRP α in the context of the innate immune system, CD47 functions as an own Marker by transmitting inhibitory "individual kill me" signals in conjunction with SIRP α expressed by myeloid cells such as macrophages, neutrophils and dendritic cells, thus CD47, expressed widely in physiological situations, serves to protect healthy cells from being eliminated by the innate immune system (Oldenborg PA, et al, CD 47-nal Regulation Protein α (sip α) regulations Fc gamma and complementary vector-derived genes, J.exp.2001. Appr 2; 7. 193-62; map Fc gamma. and 12, gene expression of Cell 19. 12, J.2001. 7. 12. of Cell 855, III. 35. 12. 7. J.7. expression of nucleic acids, III. 10. 7. 9. Cell 35. 9. origin. 10. 9. origin. A. 7. expression of nucleic acids, A. 12, A. 7. A. 7. A. 7. A. 15. biological assay, 7. A. 15. a.

Tumor cells hijack this immunosuppressive mechanism by overexpressing CD47, which effectively helps them escape immune surveillance and be killed by innate immune cells (Majeti R, Ch et al, CD47 is an adover protective factor and Therapeutic anti-Cancer target on human animal myoloid leid leukemia cells, cell 2009 Jul 23;138(2):286-99; S. Jaiswal et al, CD47 is an upregulated cytotoxic chemoloid cells and leukemia cells, avian cytocalcifications, cell 2009 Jul 23;138(2):271-85)) CD47 expression in most human cancers (e.g., those associated with NHL, AML, breast Cancer, glioma, bladder Cancer, prostate Cancer, and prostate Cancer), and The like, (CD 15, 18-14) Cancer, prostate Cancer, 85; Cancer of prostate Cancer, liver Cancer, prostate Cancer, 23; 2) 3-14, 23; prostate Cancer 99-85; Cancer of human liver Cancer, kidney, prostate Cancer, 11-32; prostate Cancer, 11-14, 11-85) and The like, (A15, 11-20, 11-85).

The widespread expression of CD47 in healthy tissue raises the issue of therapeutic safety and efficacy in that, firstly, targeting CD47 with neutralizing monoclonal Antibodies (Mab) can affect healthy tissue, leading to severe toxicity as shown in preclinical studies with mice and cynomolgus monkeys (Willingham SB, et al, Proc Natl Acad Sci U S a. 2012 Apr 24;109(17):6662-7; weiskorf K, et al, Engineered SIRP α Variants as immunethereutical addjuvants to Antibodies, science 2013 Jul 5;341(6141):88-91), and secondly, even by using alternative forms, can avoid or mitigate the mediated severe toxicity (weiskpf K, et al, science.2013 (Jul 5; 341: 6141) CD 88-91), the widespread expression of CD47 leading to rapid elimination of the target-mediated drug-mediated potency via rapid pharmacokinetic distribution, 47-mediated drug binding, and decreased potency.

Mesothelin (MSLN) is a 40 kDa Glycosylphosphatidylinositol (GPI) -linked cell surface glycoprotein that is produced by proteolysis of a 71kDa precursor. In normal tissues, mesothelin-at relatively low levels-is expressed only in mesothelial cells lining serosa such as pleura, peritoneum and pericardium. The normal physiological function of mesothelin is still unclear, but it appears to be necessary because mesothelin-deficient mice grow and reproduce normally and exhibit no apparent abnormalities.

Mesothelin is highly expressed in several types of solid tumors, such as malignant mesothelioma, ovarian cancer, pancreatic adenocarcinoma, lung adenocarcinoma, and endometrial carcinoma, biliary tract gastric cancer, and prostate cancer, as compared to normal tissue. Tumor mesothelin expression has often been associated with increased tumor invasiveness and poor clinical outcomes. The binding of mesothelin to the ovarian cancer antigen MUC16 (CA-125) has been shown to mediate cell-to-cell adhesion, possibly contributing to metastatic dissemination. In addition, mesothelin-mediated intracellular signaling has been reported to promote tumor cell proliferation, as well as resistance to chemotherapy and anoikis (programmed cell death resulting from loss of normal cell-matrix interactions).

Like most other GPI-anchored proteins, mesothelin is shed from the membrane and soluble mesothelin has been reported in the serum of tumor patients. Soluble mesothelin is therefore a useful biomarker for diagnosing mesothelin-positive tumors, and for monitoring disease progression and response to therapy. Soluble mesothelin is also considered a negative prognostic biomarker for patients with ovarian, lung or pancreatic adenocarcinoma, as well as triple negative breast cancer. Last but not least, serum mesothelin levels are predictive biomarkers in mesothelioma, as they have been found to correlate positively with the therapeutic response to mesothelin-targeted therapies.

Most tumor-associated antigens used for therapeutic targeting of solid tumors are also expressed in essential normal tissues. In contrast, mesothelin expression is usually low level and restricted to mesothelial cells (which seems to be essential). On the other hand, the cell surface expression of mesothelin is high in many solid tumors, which makes mesothelin a particularly attractive target for therapeutic intervention. Thus, a number of mesothelin-directed therapies using monoclonal antibodies, recombinant immunotoxins, antibody-drug conjugates, cancer vaccines and chimeric antigen receptor T cells are currently being developed (including clinical assessments of MPM and pancreatic adenocarcinoma in advanced trials).

The invention also provides bispecific antibodies that recognize CD47 and mesothelin.

In some embodiments, the bispecific antibody comprises a first arm that binds CD47 and a second arm that binds mesothelin, wherein the second arm binds mesothelin with high affinity and the first arm binds CD47 with low affinity, i.e., affinity sufficient to inhibit CD47/SIRP α when mesothelin is co-linked, this design allows the bispecific antibody of the invention to preferentially inhibit CD47 in normal cells, in the examples provided herein, with low affinity binding to CD 3656, and to selectively inhibit the binding of the first arm to MSLN, the bispecific antibody having low affinity to inhibit CD 3647, in addition to the bispecific antibody binding to the second arm of the bispecific antibody, which is capable of binding msxln 47, which is capable of killing msxln cells in a form as described herein, and which is capable of killing msxln antibodies in a form that is capable of targeting msxln 47, such as the bispecific antibody binding to msxln 47, and the bispecific antibody of binding to msxln 47, which is capable of targeting msxln 47, preferably, as described herein, in the bispecific antibody binding to CD 3647, and/SIRP α.

Exemplary bispecific antibodies of the invention in which at least one binding site is specific for CD47 and the second binding site is specific for mesothelin include, for example, bispecific antibodies in which the first arm comprises A5 A3 antibody, A5 A3M4 antibody, A5 A3M3 antibody, A5 A3M5 antibody, a KE8 antibody, a KE8-P6H5 antibody (also referred to herein as KE8H5), a KE8-P3B2 antibody (also referred to herein as KE8B2), a KE8-P2a2 antibody (also referred to herein as KE8a 2), a KE8F2 antibody, a KE8G2 antibody, a KE84G 2 antibody, a KE81 a2 antibody, a KE8E 2 antibody, a KE8G2 antibody, a KE8H 2 antibody, a KE8C 2, a KA 72 a KE 72 a KA 2 antibody, a KE 3a 2B 2 antibody, a 2B 2, a2, KA3H8 antibody, KA3B2 antibody, KA3a2 antibody, KA3D3 antibody, KA3H3 antibody, KC 3-P1G 11KC 3-P4C 3 antibody, KC 3-P6B 1KC 3-P4F 3 antibody, and KC 3-P2E 3 antibody (also referred to herein as KC4E 3), KC 3 antibody, KC4F 3 antibody, KC4 A3 antibody, KC4C3 antibody, KC4E 3 antibody, KC4B 3 antibody, KC4C3 antibody, KC4 A3 antibody, KC4G 3 antibody, or KC4G 3 antibody and immunologically active and/or antigen-binding fragments thereof, and wherein the second arm comprises O3 antibody, O3, O3 A3 antibody, O3, O3G 3 antibody, O3 antibody, an immunologically active and/or an antigen-binding antibody and/or an immunologically active 3 antibody and/or an antigen-binding antibody.

In some embodiments, an exemplary bispecific antibody of the invention comprising at least a first arm that binds CD47 comprises a combination of heavy and light chain Complementarity Determining Regions (CDRs) selected from the CDR sequences set forth in tables 1, 2, and 3, wherein the CDRs shown in tables 1, 2, and 3 are determined according to IMGT nomenclature.

In some embodiments, an exemplary bispecific antibody of the invention comprising at least a first arm that binds CD47 comprises a combination of a heavy chain CDR sequence from table 1 and two sets of light chain CDRs selected from the CDRL1, CDRL2 and CDRL3 sequences set forth in tables 2 and 3.

In some embodiments, an exemplary bispecific antibody of the invention comprising at least a first arm that binds CD47 comprises a combination of a heavy chain CDR sequence from table 1 and a first set of light chain CDRs selected from the group consisting of CDRL1, CDRL2, and CDRL3 sequences set forth in table 2 and a second set of light chain CDRs selected from the group consisting of CDRL1, CDRL2, and CDRL3 sequences set forth in table 3.

In some embodiments, an exemplary bispecific antibody of the invention comprises a first arm that binds CD47 and a second arm that binds MSLN, wherein the first arm comprises a combination of heavy chain Complementarity Determining Regions (CDRs) shown in table 1 and a combination of light chain CDRs selected from the CDR sequences shown in table 2, and wherein the second arm comprises a combination of heavy chain Complementarity Determining Regions (CDRs) shown in table 1 and a combination of light chain CDRs selected from the CDR sequences shown in table 4.

Table 1: common heavy chain CDR

Table 2: anti-CD 47 kappa light chain CDR

Table 3: anti-CD 47 lambda light chain CDR

Table 4: anti-CD 47 lambda light chain CDR

Each of the exemplary anti-CD 47, anti-MSLN monovalent and bispecific antibodies described herein includes a universal Heavy Chain (HC), one kappa chain and one lambda chain of an anti-CD 47 and anti-MSLN antibody, one kappa and one lambda Light Chain (LC) of a monovalent and bispecific antibody, as set forth in the amino acids and corresponding nucleic acid sequences listed below. Each of the exemplary anti-CD 47, anti-MSLN monovalent and bispecific antibodies described below includes a universal variable heavy chain (VH), one kappa variable light domain and one lambda variable light domain of an anti-CD 47 and anti-MSLN antibody, one kappa and one lambda variable light domain (VL) of a monovalent and bispecific antibody, as set forth in the amino acids and corresponding nucleic acid sequences listed below.

Although the antibody sequences below are provided herein as examples, it will be understood that these sequences may be used to generate bispecific antibodies using a variety of art-recognized techniques, examples of bispecific formats include, but are not limited to, bispecific IgG based on Fab arm exchange (Graner et al, 2013 MAbs.5(6)), CrossMab formats (Klein C et al, 2012MAbs 4(6)), various formats based on forced heterodimerization methods such as SEED techniques (Davis JH et al, 2010Protein Eng Des. 23(4): 195) 202), electrostatic manipulation (Gunasekaran K et al, J Biol Chem.2010285 (25): 19637-46) or incorporation into holes (knob-into-hole) (Ridgway JB J et al, ProteinEng. 19969 (7): 715-21) or other sets of mutations that prevent homodimer formation (Biodame.7: 31: 19: 11) or other sets of bispecific antibodies (Biodame-into-hol-ne Polyp III) such as Biodame J ü et al, (7: 35, Biodame J ü E et al, (7: 35: 11 et al, (Biodame et al; bispecific antibodies) (Biodame et al; Bignt J biochar K et al; Biodame et al; Biotech. 31; Bn III) 19, Sp 7 et al, (75; Bingwa 75; Bignt J biochar K7 et al; bispecific antibodies, Sp 7, Sp 7. 35; Bignt., Sp et al; Bignt J.

Exemplary anti-CD 47, anti-MSLN monovalent and bispecific antibodies include a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO: 1.

The anti-CD 47, anti-MSLN monovalent and bispecific antibody comprises a universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO: 113.

anti-CD 47 antibodies

The 5A3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:4) encoded by the nucleic acid sequence shown in SEQ ID NO: 3.

The 5A3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:116) encoded by the nucleic acid sequence shown in SEQ ID NO: 115.

The 5A3-M4 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:6) encoded by the nucleic acid sequence shown in SEQ ID NO: 5.

The 5A3-M4 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:118) encoded by the nucleic acid sequence shown in SEQ ID NO: 117.

The 5A3-M3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:8) encoded by the nucleic acid sequence shown in SEQ ID NO: 7.

The 5A3-M3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:120) encoded by the nucleic acid sequence shown in SEQ ID NO: 119.

The 5A3-M5 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:10) encoded by the nucleic acid sequence shown in SEQ ID NO: 9.

The 5A3-M5 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:122) encoded by the nucleic acid sequence shown in SEQ ID NO: 121.

The Ke8 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:12) encoded by the nucleic acid sequence shown in SEQ ID NO: 11.

The Ke8 antibody included the universal variable heavy domain encoded by the nucleic acid sequence shown in SEQ ID NO 113 (SEQ ID NO:114) and included the kappa variable light domain encoded by the nucleic acid sequence shown in SEQ ID NO:123 (SEQ ID NO: 124).

The Ke8H5 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:14) encoded by the nucleic acid sequence shown in SEQ ID NO: 13.

The Ke8H5 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:126) encoded by the nucleic acid sequence shown in SEQ ID NO: 125.

The Ke8B2 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:16) encoded by the nucleic acid sequence shown in SEQ ID NO: 15.

The Ke8B2 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:128) encoded by the nucleic acid sequence shown in SEQ ID NO: 127.

The Ke8A2 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:18) encoded by the nucleic acid sequence shown in SEQ ID NO: 17.

The Ke8A2 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:130) encoded by the nucleic acid sequence shown in SEQ ID NO: 129.

The Ke8E8 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:20) encoded by the nucleic acid sequence shown in SEQ ID NO: 19.

The Ke8E8 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:132) encoded by the nucleic acid sequence shown in SEQ ID NO: 131.

The Ke8H3 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:22) encoded by the nucleic acid sequence shown in SEQ ID NO: 21.

The Ke8H3 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:134) encoded by the nucleic acid sequence shown in SEQ ID NO: 133.

The Ke8G6 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:24) encoded by the nucleic acid sequence shown in SEQ ID NO: 23.

The Ke8G6 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:136) encoded by the nucleic acid sequence shown in SEQ ID NO: 135.

The Ke8A3 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:26) encoded by the nucleic acid sequence shown in SEQ ID NO: 25.

The Ke8A3 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:138) encoded by the nucleic acid sequence shown in SEQ ID NO: 137.

The Ke81A3 antibody included the universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included the kappa light chain (SEQ ID NO:28) encoded by the nucleic acid sequence shown in SEQ ID NO: 27.

The Ke81A3 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:140) encoded by the nucleic acid sequence shown in SEQ ID NO: 139.

The Ke8A8 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:30) encoded by the nucleic acid sequence shown in SEQ ID NO: 29.

The Ke8A8 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:142) encoded by the nucleic acid sequence shown in SEQ ID NO: 141.

The Ke8C7 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:32) encoded by the nucleic acid sequence shown in SEQ ID NO: 31.

The Ke8C7 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:144) encoded by the nucleic acid sequence shown in SEQ ID NO: 143.

The Ke8G2 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:34) encoded by the nucleic acid sequence shown in SEQ ID NO: 33.

The Ke8G2 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:146) encoded by the nucleic acid sequence shown in SEQ ID NO: 145.

The Ke81G9 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:36) encoded by the nucleic acid sequence shown in SEQ ID NO: 35.

The Ke81G9 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:148) encoded by the nucleic acid sequence shown in SEQ ID NO: 147.

The Ke8F2 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:38) encoded by the nucleic acid sequence shown in SEQ ID NO: 37.

The Ke8F2 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:150) encoded by the nucleic acid sequence shown in SEQ ID NO: 149.

The Ke8B7 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:40) encoded by the nucleic acid sequence shown in SEQ ID NO: 39.

The Ke8B7 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:152) encoded by the nucleic acid sequence shown in SEQ ID NO: 151.

The Ke8C4 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:42) encoded by the nucleic acid sequence shown in SEQ ID NO: 41.

The Ke8C4 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:154) encoded by the nucleic acid sequence shown in SEQ ID NO: 153.

The Ke8F1 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:44) encoded by the nucleic acid sequence shown in SEQ ID NO: 43.

The Ke8F1 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:156) encoded by the nucleic acid sequence shown in SEQ ID NO: 155.

The Ke8G11 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:46) encoded by the nucleic acid sequence shown in SEQ ID NO: 45.

The Ke8G11 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:158) encoded by the nucleic acid sequence shown in SEQ ID NO: 157.

The Ke8H6 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:48) encoded by the nucleic acid sequence shown in SEQ ID NO: 47.

The Ke8H6 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:160) encoded by the nucleic acid sequence shown in SEQ ID NO: 159.

The Ke84G9 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:50) encoded by the nucleic acid sequence shown in SEQ ID NO: 49.

The Ke84G9 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:162) encoded by the nucleic acid sequence shown in SEQ ID NO: 161.

The Ke8A4 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:52) encoded by the nucleic acid sequence shown in SEQ ID NO: 51.

The Ke8A4 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:164) encoded by the nucleic acid sequence shown in SEQ ID NO: 163.

The Ke86G9 antibody included a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and included a kappa light chain (SEQ ID NO:54) encoded by the nucleic acid sequence shown in SEQ ID NO: 53.

The Ke86G9 antibody included the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and included the kappa variable light domain (SEQ ID NO:166) encoded by the nucleic acid sequence shown in SEQ ID NO: 165.

The Ka3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:56) encoded by the nucleic acid sequence shown in SEQ ID NO: 55.

The Ka3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:168) encoded by the nucleic acid sequence shown in SEQ ID NO: 167.

The Ka3A2 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:58) encoded by the nucleic acid sequence shown in SEQ ID NO: 57.

The Ka3A2 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:170) encoded by the nucleic acid sequence shown in SEQ ID NO: 169.

The Ka3H3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:60) encoded by the nucleic acid sequence shown in SEQ ID NO: 59.

The Ka3H3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:172) encoded by the nucleic acid sequence shown in SEQ ID NO: 171.

The Ka3A3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:62) encoded by the nucleic acid sequence shown in SEQ ID NO: 61.

The Ka3A3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:174) encoded by the nucleic acid sequence shown in SEQ ID NO: 173.

The Ka3H8 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:64) encoded by the nucleic acid sequence shown in SEQ ID NO: 63.

The Ka3H8 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:176) encoded by the nucleic acid sequence shown in SEQ ID NO: 175.

The Ka3B2 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:66) encoded by the nucleic acid sequence shown in SEQ ID NO: 65.

The Ka3B2 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:178) encoded by the nucleic acid sequence shown in SEQ ID NO: 177.

The Ka3C5 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:68) encoded by the nucleic acid sequence shown in SEQ ID NO: 67.

The Ka3C5 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:180) encoded by the nucleic acid sequence shown in SEQ ID NO: 179.

The Ka3G2 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:70) encoded by the nucleic acid sequence shown in SEQ ID NO: 69.

The Ka3G2 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:182) encoded by the nucleic acid sequence shown in SEQ ID NO: 181.

The Ka3D3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a kappa light chain (SEQ ID NO:72) encoded by the nucleic acid sequence shown in SEQ ID NO: 71.

The Ka3D3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the kappa variable light domain (SEQ ID NO:184) encoded by the nucleic acid sequence shown in SEQ ID NO: 183.

The Kc4 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:74) encoded by the nucleic acid sequence shown in SEQ ID NO: 73.

The Kc4 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:186) encoded by the nucleic acid sequence shown in SEQ ID NO: 185.

The Kc4G11 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:76) encoded by the nucleic acid sequence shown in SEQ ID NO: 75.

The Kc4G11 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:188) encoded by the nucleic acid sequence shown in SEQ ID NO: 187.

The Kc4C11 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:78) encoded by the nucleic acid sequence shown in SEQ ID NO: 77.

The Kc4C11 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:190) encoded by the nucleic acid sequence shown in SEQ ID NO: 189.

The Kc4A1 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:80) encoded by the nucleic acid sequence shown in SEQ ID NO: 79.

The Kc4A1 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:192) encoded by the nucleic acid sequence shown in SEQ ID NO: 191.

The Kc4A4 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:82) encoded by the nucleic acid sequence shown in SEQ ID NO: 81.

The Kc4A4 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:194) encoded by the nucleic acid sequence shown in SEQ ID NO: 193.

The Kc4E10 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:84) encoded by the nucleic acid sequence shown in SEQ ID NO: 83.

The Kc4E10 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:196) encoded by the nucleic acid sequence shown in SEQ ID NO: 195.

The Kc4G9 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:86) encoded by the nucleic acid sequence shown in SEQ ID NO: 85.

The Kc4G9 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:198) encoded by the nucleic acid sequence shown in SEQ ID NO: 197.

The Kc4C3 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:88) encoded by the nucleic acid sequence shown in SEQ ID NO: 87.

The Kc4C3 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:200) encoded by the nucleic acid sequence shown in SEQ ID NO: 199.

The Kc4F4 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:90) encoded by the nucleic acid sequence shown in SEQ ID NO: 89.

The Kc4F4 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:202) encoded by the nucleic acid sequence shown in SEQ ID NO: 201.

The Kc4B1 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:92) encoded by the nucleic acid sequence shown in SEQ ID NO: 91.

The Kc4B1 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:204) encoded by the nucleic acid sequence shown in SEQ ID NO: 203.

The Kc4E2 antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:94) encoded by the nucleic acid sequence shown in SEQ ID NO: 93.

The Kc4E2 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:96) encoded by the nucleic acid sequence shown in SEQ ID NO: 95.

Anti-mesothelin (anti-msln) antibodies

O25The antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:98) encoded by the nucleic acid sequence shown in SEQ ID NO: 97. The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O25 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:212) encoded by the nucleic acid sequence shown in SEQ ID NO: 211.

O30The antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:100) encoded by the nucleic acid sequence shown in SEQ ID NO: 99. The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O30 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:214) encoded by the nucleic acid sequence shown in SEQ ID NO: 213.

O32The antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:102) encoded by the nucleic acid sequence shown in SEQ ID NO: 101. The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O32 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:216) encoded by the nucleic acid sequence shown in SEQ ID NO: 215.

O35The antibody includes the universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes the universal heavy chain (SEQ ID NO: 103)The nucleic acid sequence shown encodes a lambda light chain (SEQ ID NO: 104). The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O35 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:218) encoded by the nucleic acid sequence shown in SEQ ID NO: 217.

O37The antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:106) encoded by the nucleic acid sequence shown in SEQ ID NO: 105. The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O37 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:220) encoded by the nucleic acid sequence shown in SEQ ID NO: 219.

O38The antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:108) encoded by the nucleic acid sequence shown in SEQ ID NO: 107. The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O38 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:222) encoded by the nucleic acid sequence shown in SEQ ID NO: 221.

O41The antibody includes a universal heavy chain (SEQ ID NO:2) encoded by the nucleic acid sequence shown in SEQ ID NO:1 and includes a lambda light chain (SEQ ID NO:110) encoded by the nucleic acid sequence shown in SEQ ID NO: 109. The variable region of the lambda light chain is in bold in the amino acid sequence below.

The O41 antibody includes the universal variable heavy domain (SEQ ID NO:114) encoded by the nucleic acid sequence shown in SEQ ID NO:113 and includes the lambda variable light domain (SEQ ID NO:224) encoded by the nucleic acid sequence shown in SEQ ID NO: 223.

Pseudolight chains

Pseudolight chain 1 (SEQ ID NO:112) is encoded by the nucleic acid sequence shown in SEQ ID NO: 111.

Pseudo variable light chain domain 1 (SEQ ID NO:206) is encoded by the nucleic acid sequence shown in SEQ ID NO: 205.

Pseudo light chain 2 (SEQ ID NO:208) is encoded by the nucleic acid sequence shown in SEQ ID NO: 207.

Pseudo variable light chain domain 2 (SEQ ID NO:210) is encoded by the nucleic acid sequence shown in SEQ ID NO: 209.

Bispecific antibodies

In some embodiments, bispecific antibody Ka3 x O25 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:283, CDRL2 comprising the amino acid sequence of SEQ ID NO:289 and a lambda light chain comprising CDRL3 comprising the amino acid sequence of SEQ ID NO: 295.

In some embodiments, bispecific antibody Ka3 x O25 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:211 (SEQ ID NO: 212).

In some embodiments, bispecific antibody Ka3 x O25 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:97 (SEQ ID NO: 98).

In some embodiments, bispecific antibody Ka3 x O30 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:284, CDRL2 comprising the amino acid sequence of SEQ ID NO:290, and cdl 3 comprising the amino acid sequence of SEQ ID NO: 296.

In some embodiments, bispecific antibody Ka3 x O30 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:213 (SEQ ID NO: 214).

In some embodiments, bispecific antibody Ka3 x O30 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:99 (SEQ ID NO: 100).

In some embodiments, bispecific antibody Ka3 x O32 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:282, CDRL2 comprising the amino acid sequence of SEQ ID NO:288, and cdl 3 comprising the amino acid sequence of SEQ ID NO: 294.

In some embodiments, bispecific antibody Ka3 x O32 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:215 (SEQ ID NO: 216).

In some embodiments, bispecific antibody Ka3 x O32 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:101 (SEQ ID NO: 102).

In some embodiments, bispecific antibody Ka3 x O35 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a CDRL1 comprising the amino acid sequence of SEQ ID NO:287, CDRL2 comprising the amino acid sequence of SEQ ID NO:293, and a lambda light chain comprising cdl 3 comprising the amino acid sequence of SEQ ID NO: 299.

In some embodiments, bispecific antibody Ka3 x O35 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:217 (SEQ ID NO: 218).

In some embodiments, bispecific antibody Ka3 x O35 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:103 (SEQ ID NO: 104).

In some embodiments, bispecific antibody Ka3 x O37 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a CDRL1 comprising the amino acid sequence of SEQ ID NO:285, CDRL2 comprising the amino acid sequence of SEQ ID NO:291, and a lambda light chain comprising cdl 3 comprising the amino acid sequence of SEQ ID NO: 297.

In some embodiments, bispecific antibody Ka3 x O37 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:219 (SEQ ID NO: 220).

In some embodiments, bispecific antibody Ka3 x O37 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:105 (SEQ ID NO: 106).

In some embodiments, bispecific antibody Ka3 x O38 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:286, CDRL2 comprising the amino acid sequence of SEQ ID NO:292, and cdl 3 comprising the amino acid sequence of SEQ ID NO: 298.

In some embodiments, bispecific antibody Ka3 x O38 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:221 (SEQ ID NO: 222).

In some embodiments, bispecific antibody Ka3 x O38 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:109 (SEQ ID NO: 108).

In some embodiments, bispecific antibody Ka3 x O41 comprises a common heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242, and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:282, CDRL2 comprising the amino acid sequence of SEQ ID NO:288, and cdl 3 comprising the amino acid sequence of SEQ ID NO: 300.

In some embodiments, bispecific antibody Ka3 x O41 includes a common heavy chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:113 (SEQ ID NO:114), a kappa light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:167 (SEQ ID NO:168), and a lambda light chain variable region encoded by the nucleic acid sequence shown in SEQ ID NO:223 (SEQ ID NO: 224).

In some embodiments, bispecific antibody Ka3 x O41 includes a common heavy chain encoded by the nucleic acid sequence shown in SEQ ID NO:1 (SEQ ID NO:2), a kappa light chain encoded by the nucleic acid sequence shown in SEQ ID NO:55 (SEQ ID NO:56), and a lambda light chain encoded by the nucleic acid sequence shown in SEQ ID NO:111 (SEQ ID NO: 112).

Defining:

unless defined otherwise, technical and scientific terms used in the context of the present invention have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall also include the plural and plural terms shall also include the singular. Generally, the nomenclature used and the techniques described herein in connection with, cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry and hybridization are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques were performed according to manufacturer's instructions, or as commonly performed in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al Molecular Cloning: A LABORATORY MANUAL (second edition, Cold Spring Harbor LABORATORY Press, Cold Spring Harbor, N.Y. (1989)). The terminology used in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

As used in accordance with the present invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

as used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds to (immunoreacts with) an antigen. By "specifically binds" or "immunoreactive with … …" or "immunospecific binding" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or with much lower affinity (K)_d>10^-6) And (4) combining. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, F_ab、F_ab'And F_(ab')2Fragments, scFVs, and F_abAn expression library.

Basic antibody building blocks are known to comprise tetramers. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The amino terminus of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Generally, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other due to the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG₁、IgG₂And others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

The term "monoclonal antibody (MAb)" or "monoclonal antibody composition" as used herein refers to a population of antibody molecules containing only one molecular species of antibody molecules consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the Complementarity Determining Regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. Mabs contain antigen binding sites that are capable of immunoreacting with a particular epitope of an antigen characterized by unique binding affinity for it.

The term "antigen binding site" or "binding portion" refers to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sequences within the V regions of the heavy and light chains, termed "hypervariable regions", are located between the more conserved flanking sequences termed "framework regions" or "FRs". Thus, the term "FR" refers to amino acid sequences that are naturally located between and adjacent to hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs". The amino acid assignment to each domain is consistent with the definition of Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk J. mol. biol. 196: 901. 917 (1987), Chothia et al Nature 342: 878. snake 883 (1989).

As used herein, the term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T cell receptor. The term "epitope" includes any protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. Epitopic determinants are typically composed of molecules of chemically active surface components (such as amino acids or sugar side chains) and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, antibodies can be raised against the N-terminal or C-terminal peptide of a polypeptide. When the dissociation constant is less than or equal to 1 mu M; for example 100 nM or less, preferably 10 nM or less and more preferably 1 nM or less, the antibody binds specifically to the antigen.

As used herein, the terms "immunological binding" and "immunological binding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction may be the dissociation constant (K) of the interaction_d) Is expressed in the form of (1), wherein the smaller K_dIndicating greater affinity. The immunological binding properties of the selected polypeptide can be quantified using methods well known in the art. One such method entails measuring the rates of antigen binding site/antigen complex formation and dissociation, where those rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that also affect the rate in both directions. Thus, the "association rate constant" (K)_on) And an "off rate constant" (K)_off) Can be determined by calculating the concentration and the actual rate of binding and dissociation. (see Nature 361:186-87 (1993)). K_off/K_onAllows to delete all the parameters not related to affinity and is equal to the dissociation constant K_d. (see generally Davies et al (1990) Annual RevBiochem 59: 439-. When equilibrium binding constant (K) as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art_d) An antibody of the invention specifically binds its target at ≦ 1 μ M, e.g., ≦ 100 nM, preferably ≦ 10 nM, and more preferably ≦ 1 nM.

The term "isolated polynucleotide" as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, by virtue of its origin, the "isolated polynucleotide" (1) does not accompany all or a portion of the polynucleotide in which it is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not exist in nature as part of a larger sequence. Polynucleotides according to the invention include nucleic acid molecules encoding heavy chain immunoglobulin molecules and nucleic acid molecules encoding light chain immunoglobulin molecules as described herein.

The term "isolated protein" refers herein to a cDNA, recombinant RNA, or protein of synthetic origin, or some combination thereof, which, due to its origin or derived source (source of derivation), the "isolated protein" (1) does not accompany a protein found in nature, (2) does not contain other proteins from the same source, e.g., does not contain marine proteins, (3) is expressed by a different kind of cell, or (4) does not occur in nature.

The term "polypeptide" is used herein as a generic term to refer to a native protein, fragment, or analog of a polypeptide sequence. Thus, native protein fragments and analogs are species (species) of the genus Polypeptides (genus). Polypeptides according to the invention include heavy and light chain immunoglobulin molecules as described herein, as well as antibody molecules formed from combinations comprising heavy and light chain immunoglobulin molecules (such as kappa light chain immunoglobulin molecules, and vice versa), fragments and analogs thereof.

The term "naturally-occurring" as used herein applies to an object to refer to the fact that the object is discoverable in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been purposefully altered in the laboratory by man or otherwise is naturally occurring.

The term "operably linked" as used herein refers to the positions of the components in a relationship that allows them to function in their intended manner. A control sequence is "operably linked" to a coding sequence in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term "control sequences" as used herein refers to polynucleotide sequences necessary to effect expression and processing of the coding sequences to which they are ligated. The nature of such control sequences varies depending on the host organism, and in prokaryotes such control sequences generally include a promoter, a ribosome binding site and a transcription termination sequence, and in eukaryotes such control sequences generally include a promoter and a transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. The term "polynucleotide" as referred to herein means a polymeric boron (polymeric boron) of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or modified forms of either type of nucleotide. The term includes single-stranded and double-stranded forms of DNA.

As used herein, twenty common amino acids and their abbreviations are according to conventional usage, see Immunology-ASynthesis (second edition, e.s. gold and d.r.gren, eds., Sinauer Associates, Sunderland mass. (1991)), stereoisomers of twenty common amino acids (e.g., D-amino acids), unnatural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unusual amino acids may also be suitable components of the polypeptides of the invention.

The term "substantial identity" when applied to a polypeptide means that two peptide sequences have at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity when optimally aligned by using default GAP weights, such as the programs GAP or BESTFIT.

Preferably, residue positions that are not identical differ by conservative amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; one group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; one group of amino acids with aromatic side chains is phenylalanine, tyrosine and tryptophan; one group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

As discussed herein, minor changes in the amino acid sequences of antibodies and immunoglobulin molecules are understood to be encompassed by the present invention provided that the changes in the amino acid sequences remain at least 75%, preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those substitutions that occur within a family of amino acids related to their side chain. Genetically encoded amino acids are generally classified into the following families: (1) the acidic amino acid is aspartic acid or glutamic acid; (2) the basic amino acid is lysine, arginine and histidine; (3) the nonpolar amino acid is alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) the uncharged polar amino acids are glycine, asparagine, glutamine; cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) arginine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan and tyrosine, which are aromatic families. For example, it is reasonably expected that the substitution of a leucine alone with an isoleucine or valine, an aspartic acid alone with a glutamic acid, a threonine alone with a serine, or an amino acid similarly with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can be readily determined by determining the specific activity of the polypeptide derivative. The assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by one of ordinary skill in the art. Fragments or analogs preferably have the amino-terminal and carboxy-terminal ends present near the boundaries of the functional domain. Structural and functional domains can be identified by comparing nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains in other proteins that now have known structure and/or function. Methods for identifying protein sequences that fold into a known three-dimensional structure are known. Bowie et al Science 253:164 (1991). Thus, the above examples demonstrate that one skilled in the art can identify sequence motifs and structural conformations that can be used to define the structural and functional domains of the invention.

Preferred amino acid substitutions are those which are: (1) reducing susceptibility to proteolysis, (2) reducing susceptibility to oxidation, (3) altering binding affinity for the protein complex formed, (4) altering binding affinity, and (4) conferring or modifying other physicochemical or functional properties to such analogs. Analogs can include various muteins of a sequence other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably, conservative amino acid substitutions) may be made in the naturally occurring sequence (preferably, in the part of the polypeptide outside of the domains forming intermolecular contacts). Conservative amino acid substitutions should not substantially alter the structural characteristics of the parent sequence (e.g., the substituted amino acid should tend not to break a helix formed in the parent sequence, or disrupt other types of secondary structure that is characteristic of the parent sequence). Examples of secondary and tertiary Structures of polypeptides recognized in the art are described in Proteins, Structures and molecular Principles (Creighton, ed., W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (C. Brandn and J. Tooze, ed., Garland Publishing, New York, N.Y. (1991)), and Thornton et al Nature 354: 105: 1991).

As used herein, the term "label" or "labeled" refers to incorporation of a detectable label, e.g., by incorporation of a radiolabeled amino acid or a polypeptide linked to a biotin-based moiety that can be detected by labeled avidin (e.g., streptavidin containing a fluorescent label or enzymatic activity that can be detected by optical or calorimetric methods). In some cases, the markers and markers may also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, the following: a radioisotope or radionuclide (e.g.,³H、¹⁴C、¹⁵N、³⁵S、⁹⁰Y、⁹⁹Tc、¹¹¹In、¹²⁵I、¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labels are linked by spacer arms of various lengths to reduce potential steric hindrance. The term "agent or drug" as used herein refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

Other Chemical Terms are used herein according to conventional usage in The art, as exemplified by The McGraw-Hilldictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

As used herein, "substantially pure" means that the target species is the predominant species present (i.e., it is more abundant on a molar basis than any other individual species in the composition), and preferably that the substantially purified fraction is a composition in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present.

Generally, a substantially pure composition will contain more than about 80% of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95% and 99%. Most preferably, the target species is purified to substantial homogeneity (contaminant species are not detectable in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

The term patient includes human and veterinary subjects (veterinary subjects).

Antibodies

Various procedures known in the art can be used to generate polyclonal or monoclonal antibodies against a given target, e.g., CD47, a tumor-associated antigen or other target, or against derivatives, fragments, analogs, homologs or orthologs thereof. (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

Antibodies are purified by well-known techniques such as affinity chromatography using protein a or protein G, which provides primarily the IgG fraction of the immune serum. Subsequently, or alternatively, an antigen or epitope thereof specific for the target of the immunoglobulin sought may be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed for example by d.wilkinson (The Scientist, published by The Scientist, inc., philiadelphia pa, vol. 14, number 8 (April 17, 2000), pp. 25-28).

In some embodiments, the antibodies of the invention may be monoclonal antibodies. Monoclonal antibodies are generated, for example, by using the procedures described in the examples provided herein. Antibodies are also generated, for example, by immunizing BALB/c mice with a combination of transfectants of cells expressing high levels of a given target on their surface. Hybridomas produced by myeloma/B cell fusions are then screened for reactivity against the selected target.

Monoclonal antibodies are prepared, for example, using hybridoma methods such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

The immunizing agent will typically include a protein antigen, fragment thereof, or fusion protein thereof. Typically, peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian origin is desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding,Monoclonal Antibodies:Principles and Practiceacademic Press, (1986) pp. 59-103). Immortalized cell lines are generally transformed with mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium of the hybridoma will contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. A more preferred immortalized Cell line is a murine myeloma line, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies. (see Kozbor, J. Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by Scatchard analysis of Munson and Pollard, anal. biochem., 107:220 (1980). Furthermore, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (see also the Goding of the fact that,Monoclonal Antibodies:Principles and Practiceacademycpress, (1986) pp. 59-103). Suitable media for this purpose include, for example, Dulbecco's modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo in a mammal as ascites fluid.

Monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods such as those described in U.S. patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is subsequently transfected into a host cell, such as a simian COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulin protein, to obtain synthesis of monoclonal antibodies in the recombinant host cell. DNA may also be modified, for example, by replacing the homologous murine sequences with the coding sequences for the human heavy and light chain constant domains (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking to the coding sequence of a non-immunoglobulin polypeptide all or part of the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides may replace the constant domains of an antibody of the invention, or may replace the variable domains of one antigen binding site of an antibody of the invention, to produce a chimeric bivalent antibody.

The monoclonal antibody of the present invention includes a humanized antibody or a human antibody. These antibodies are suitable for administration to humans without eliciting an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof comprising predominantly the sequence of a human immunoglobulin and containing minimal sequence derived from a non-human immunoglobulin (such as Fv, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). Humanization is carried out, for example, by replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences according to the method of Winter and coworkers (Jones et al, Nature, 321:522-525 (1986); Riechmann et al, Nature, 332:323-327 (1988); Verhoeyen et al, Science, 239:1534-1536 (1988)). (see also U.S. Pat. No. 5,225,539). In some cases, Fv framework residues of the human immunoglobulin are substituted with corresponding non-human residues. Humanized antibodies also comprise residues that are not found, for example, in the recipient antibody or in imported CDR or framework sequences. Typically, a humanized antibody comprises substantially all, at least one, and typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody also most preferably comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al, 1986; Riechmann et al, 1988; and Presta, curr. Op. struct. biol., 2:593-596 (1992)).

Fully human antibodies are antibody molecules in which the entire sequence of both the light and heavy chains, including the CDRs, are derived from a human gene. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Monoclonal antibodies can be generated by using the trioma technique; human B cell hybridoma technology (see Kozbor, et al, 1983Immunol Today 4: 72); and EBV hybridoma technology (see Cole, et al, 1985 In: Monoclonal Antibodies and cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal Antibodies are available and can be generated using human hybridomas (see Cote, et al, 1983.Proc Natl Acad Sci USA 80: 2026-.

In addition, human antibodies can also be generated using additional techniques, including phage display libraries. (see Hoogenboom and Winter, J. mol. biol., 227:381 (1991); Marks et al, J. mol. biol.,222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, such as mice, in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production was observed, which is similar to that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. Such methods are described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, and Marks et al, Bio/Technology 10, 779-783 (1992), Lonberg et al, Nature 368856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al, Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826(1996), and Lonberg and Huszar, Intern. Rev. Immunol. 1365-93 (1995).

Human antibodies can additionally be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to antigen challenge rather than animal endogenous antibodies. (see PCT publication WO 94/02602). Endogenous genes encoding the heavy and light immunoglobulin chains in non-human hosts have been disabled and active sites encoding human heavy and light chain immunoglobulins are inserted into the host genome. For example, a yeast artificial chromosome containing the necessary human DNA segments is used to incorporate the human genes. Animals provided with all desired modifications were then passed through a transgenic animal that would contain less than all of the complement of the modificationsAnimals are crossed and obtained as offspring. An example of such a non-human animal is the so-called Xenomouse as disclosed in PCT publications WO 96/33735 and WO 96/34096^TMThe mouse of (1). The animal produces B cells that secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with the immunogen of interest, e.g., as a preparation of polyclonal antibodies, or alternatively, from immortalized B cells derived from the animal, such as monoclonal antibody-producing hybridomas. In addition, the genes encoding the immunoglobulins with human variable regions may be recovered and expressed to obtain the antibodies directly, or may be further modified to obtain analogs of the antibodies, such as, for example, single chain fv (scfv) molecules.

An example of a method of producing a non-human host (exemplified by a mouse) lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Pat. No. 5,939,598. It may be obtained by a method comprising deleting J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent locus rearrangement and prevent transcript formation of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and generating from the embryonic stem cells a transgenic mouse whose somatic and germ cells both contain a gene encoding a selectable marker.

One method of producing antibodies of interest, such as human antibodies, is disclosed in U.S. patent No. 5,916,771. The method comprises introducing an expression vector comprising a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector comprising a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cells express antibodies containing heavy and light chains.

In a further improvement to this procedure, methods for identifying clinically relevant epitopes on immunogens and related methods for selecting antibodies that specifically bind the relevant epitopes with high affinity are disclosed in PCT publication WO 99/53049.

The antibody may be expressed from a vector containing a DNA segment encoding a single chain antibody as described above.

These may include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, and the like. Vectors include chemical conjugates such as those described in WO 93/64701, having a targeting moiety (e.g., a ligand for a cell surface receptor), and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT/US95/02140 (WO 95/22618), which are fusion proteins containing a target moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phage, and the like. The vector may be chromosomal, nonchromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These include pox vectors (pox vector) such as smallpox or fowlpox (orthopox or avipox) vectors, herpes virus vectors such as herpes simplex I (HSV) vectors (see Geller, A.I. et al, J. Neurochem, 64:487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A.I. et al, Proc Natl. Acad. Sci: U.S.A. 90:7603 (1993); Geller, A.I. et al, Proc Natl. Acad. Sci. et al, Proc Natl. Acad. USA 87:1149 (USA), adenovirus vectors (Legal Las et al, Sci., 8: 1993; Yaplo et al, Natl. 1990: Sal J. adenovirus vectors (1995: J. 1993) (see Gene Laplo et al, adenovirus vectors) (see Gene Laplo et al, USA, J. 1993), M.G. et al, nat. Genet. 8:148 (1994).

Poxvirus vectors introduce genes into the cytoplasm. Fowlpox viral vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and Herpes Simplex Virus (HSV) vectors are preferred for introducing nucleic acids into nerve cells. Adenovirus vectors result in shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend on the target cell and the conditions of the treatment. Introduction can be by standard techniques such as infection, transfection, transduction or transformation. Examples of patterns of gene transfer include, for example, naked DNA, CaPO₄Precipitation, DEAE-dextran, electroporation, protoplastsBody fusion, lipofection, cellular microinjection, and viral vectors.

The vector can be utilized to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to a desired location. In addition, microparticles can be delivered by using a micro-pump Infusion System such as a syncromed Infusion System intraventricular (icv) Infusion. Bulk flow based methods (known as convection) have also proven effective in delivering macromolecules to extended regions of the brain and can be used to deliver vectors to target cells. (see Bobo et al, Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al, Am. J. Physiol.266:292-305 (1994)). Other methods that may be used include catheters, intravenous, parenteral, intraperitoneal, and subcutaneous injections, and oral or other known routes of administration.

Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities is for a target such as CD47 or any fragment thereof. The second binding target is any other antigen and is advantageously a cell surface protein or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy/light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (cell hybridomas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished by an affinity chromatography step. Similar procedures are disclosed in WO93/08829, 5.13.1993, and in Traunecker et al, EMBO J., 10:3655-3659 (1991).

The bispecific and/or monovalent antibodies of the present aspects can be prepared using any of a variety of art-recognized techniques, including those disclosed in co-pending application WO 2012/023053 filed on 16/8/2011 (the contents of which are incorporated herein by reference in their entirety). The method described in WO 2012/023053 produces bispecific antibodies that are structurally identical to human immunoglobulins. Such molecules consist of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant kappa domain, and a second light chain variable region fused to a constant lambda domain. Each binding site exhibits a different antigen specificity contributed by the heavy and light chains. The light chain variable region may be of the lambda or kappa family and is preferably fused to the lambda and kappa constant domains respectively. This is preferred to avoid the production of non-native polypeptide linkers. However, bispecific antibodies of the invention may also be obtained by fusing a kappa light chain variable domain to a constant lambda domain (for the first specificity) and a lambda light chain variable domain to a constant kappa domain (for the second specificity). The bispecific antibodies described in WO 2012/023053 are referred to as IgG kappa lambda antibodies or "kappa lambda bodies", a novel fully human bispecific IgG format. This kappa lambda format allows for affinity purification of bispecific antibodies that are indistinguishable from standard IgG molecules with characteristics that are indistinguishable from standard monoclonal antibodies and therefore advantageous when compared to the previous format.

An essential step of the method is the identification of two antibody Fv regions (each consisting of a variable light chain and a variable heavy chain domain) with different antigen specificities that share the same heavy chain variable domain. Various methods have been described for generating monoclonal antibodies and fragments thereof. (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the sequences of both the light and heavy chains, including CDRs 1 and 2, are derived from human genes. The CDR3 regions may be of human origin or designed by synthetic methods. Such antibodies are referred to herein as "human antibodies" or "fully human antibodies". Human monoclonal antibodies can be generated by using a trioma technique; human B cell hybridoma technology (see Kozbor, et al, 1983Immunol Today 4: 72); and EBV hybridoma technology (see Cole, et al, 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human Monoclonal Antibodies are available and can be generated using human hybridomas (see Cote, et al, 1983.Proc Natl Acad Sci USA 80: 2026-.

For example, monoclonal antibodies are generated by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof. Alternatively, an animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding a target antigen such that the target antigen is expressed and binds to the surface of the transfected cells. Various techniques for producing xenogenic non-human animals are well known in the art. See, for example, U.S. Pat. nos. 6,075,181 and 6,150,584, which are incorporated herein by reference in their entirety.

Alternatively, the antibody is obtained by screening a library containing antibody or antigen-binding domain sequences that bind to the target antigen. The library is prepared, for example, in phage as a fusion with a phage coat protein expressed on the surface of the assembled phage particle and a protein or peptide encoding DNA sequence contained within the phage particle (i.e., a "phage display library").

Hybridomas resulting from myeloma/B cell fusions are then screened for reactivity against the target antigen. Monoclonal antibodies are prepared, for example, using hybridoma methods such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes may be immunized in vitro.

Although not strictly impossible, the accidental identification of different antibodies with the same heavy chain variable domain but directed against different antigens is highly unlikely. Indeed, in most cases, the heavy chain contributes most to the antigen binding surface and is also the most variable in sequence. Specifically, CDR3 on the heavy chain is the most diverse CDR in sequence, length, and structure. Thus, two antibodies specific for different antigens will almost certainly carry different heavy chain variable domains.

The method disclosed in co-pending application WO 2012/023053 overcomes this limitation and greatly facilitates the isolation of antibodies having identical heavy chain variable domains by using antibody libraries in which the heavy chain variable domains are identical for all library members and thus diversity is limited to light chain variable domains. Such libraries are described, for example, in co-pending applications WO 2010/135558 and WO 2011/084255, each of which is incorporated by reference herein in its entirety. However, because the light chain variable domain is co-expressed with the heavy chain variable domain, both domains may contribute to antigen binding. To further facilitate the method, antibody libraries containing the same heavy chain variable domain and diverse λ variable light chains or κ variable light chains may be used in parallel to select antibodies against different antigens in vitro. This method enables the identification of two antibodies having a common heavy chain but one carrying a lambda light chain variable domain and the other carrying a kappa light chain variable domain, which can be used as building blocks for the generation of bispecific antibodies in the full immunoglobulin format of the invention. The bispecific antibodies of the invention may be of different isotypes and their Fc portions may be modified to alter the binding properties to different Fc receptors and in this way modify the effector functions of the antibody as well as its pharmacokinetic properties. Various methods of modifying the Fc portion have been described and can be applied to the antibodies of the invention. (see, for example, Strohl, WR Curr Opin Biotechnol 2009 (6):685-91; U.S. Pat. No. 6,528,624; PCT/US2009/0191199, filed 1/9/2009, 2009). The methods of the invention can also be used to generate bispecific antibodies and antibody mixtures in the form of F (ab') 2 lacking an Fc portion.

The ratio of monospecific (same light chain) and bispecific (two different light chains) should then be 50% if all polypeptides are expressed at the same level and assembled equally well to form an immunoglobulin molecule, however, it is possible that different light chains are expressed at different levels and/or not assembled with the same efficiency.thus, methods of regulating the relative expression of different polypeptides are used to compensate for their intrinsic expression characteristics or different tendencies to assemble with the common heavy chain. this regulation can be achieved via promoter strength, using Internal Ribosome Entry Sites (IRES) characterized by different efficiencies or other types of regulatory elements (which can act on transcription or translation levels and on mRNA stability.) different promoters of different strengths can include CMV (immediate early cytomegalovirus promoter); Ubc (human C promoter); SV 36 (monkey 40 promoter) different promoters from mammalian and viral origin (human elongation factor 1 α -subunit promoter); Sa 1-1 (human IRES promoter) can also be used to regulate the relative expression of various Genes from a variety of mammalian and viruses; thus the relative expression of the various Genes can be further regulated by introducing multiple copies of the heterologous Genes-see, e.g. Severnle.g. Selen Secrena, Selen et al. (see, et al.: further, the expression of various Genes; see, et al.: it is illustrative how many times the regulation of the relative expression of the expression of various Genes expressed on the relative expression of the relevant Genes of the various Genes-Secrenell-Adenon. A-Adenon.

Co-expression of the heavy chain and two light chains produced a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter must be purified from the mixture to obtain the target molecule. The methods described herein greatly facilitate this purification procedure by using affinity chromatography media that specifically interact with kappa or lambda light chain constant domains such as CaptureSelect Fab kappa and CaptureSelect Fab lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification method is effective and generally applicable to the antibodies of the present invention. This is in contrast to the specific purification methods that must be developed and optimized for each bispecific antibody derived from a cell hybridoma or other cell line expressing a mixture of antibodies. Indeed, if the biochemical characteristics of different antibodies in a mixture are similar, their separation using standard chromatographic techniques such as ion exchange chromatography may be challenging, or not possible at all.

Other suitable purification methods include those disclosed in WO2013/088259, the contents of which are incorporated by reference in their entirety, co-pending application PCT/IB2012/003028 filed on day 19, 10/2012.

In other embodiments of generating bispecific antibodies, antibody variable domains (antibody-antigen binding sites) with the desired binding specificities can be fused to immunoglobulin constant domain sequences. The fusion preferably has an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2, and CH3 regions. Preferably, there is a first heavy chain constant region (CH1) containing a site necessary for light chain binding present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology,121:210 (1986).

According to another approach described in WO 96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces include at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). Complementary "cavities" of the same or similar size to the large side chains are created at the interface of the second antibody molecule by substituting the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism to increase the yield of heterodimers relative to other unwanted end products such as dimers.

Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical bonds. The bispecific antibodies produced can be used as reagents for the selective immobilization of enzymes.

Various techniques for the direct preparation and isolation of bispecific antibody fragments from recombinant cell cultures have also been described. For example, leucine zipper has been usedChains produce bispecific antibodies. Kostelny et al, J. Immunol.148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and subsequently re-oxidized to form antibody heterodimers. The method can also be used to produce antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA90: 6444-. The fragments comprise a light chain variable domain (V) joined by a linker_L) Connected heavy chain variable domains (V)_H) The linker is too short to allow pairing between two domains on the same strand. Thus, V of a segment_HAnd V_LThe domains are forced to complement V of another fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single chain fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol.152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies may be prepared. Tutt et al, J.Immunol.147:60 (1991).

An exemplary bispecific antibody can bind two different epitopes, at least one of which is derived from a protein antigen of the invention. Alternatively, the anti-antigen arms of immunoglobulin molecules may be combined with arms that bind to priming molecules on leukocytes, such as T cell receptor molecules (e.g., CD2, CD3, CD28, or B7) or Fc receptors of IgG (Fc γ R), such as Fc γ RI (CD64), Fc γ RII (CD32), and Fc γ RIII (CD16), to focus cellular defense mechanisms on cells expressing a particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a particular antigen. These antibodies have an antigen-binding arm and an arm that binds a cytotoxic agent or radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to a protein antigen described herein and also binds to Tissue Factor (TF).

Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (see WO 91/00360; WO 92/200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptobutyrylimidate (mercaptobutyliminate) and those disclosed, for example, in U.S. Pat. No. 4,676,980.

It may be desirable to modify the antibodies of the invention in effector function to enhance, for example, the efficacy of the antibodies in treating cancer and/or other diseases or disorders associated with aberrant CD47 expression and/or activity. For example, one or more cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus produced may have improved capacity for internalization and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (see Caron et al, J. Exp. Med., 176: 1191-. Alternatively, antibodies with dual Fc regions can be engineered and thus have enhanced complement lysis and ADCC capabilities. (see Stevenson et al, Anti-Cancer Drug Design, 3:219-230 (1989)).

The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof) or a radioisotope (i.e., a radioconjugate).

Enzymatically active toxins or fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, Lotus root toxin A chain, α sarcina, Aleurites fordii protein, Carcinia huperziana (Phytolacca americana) protein (PAPI, PAPII, and PAP-S), Momordica charantia (momordia charantia) inhibitors, and the like,Curcin, crotin, Saponaria officinalis (Sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, trichothecenes (tricothecenes). A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include²¹²Bi、¹³¹I、¹³¹In、⁹⁰Y, and¹⁸⁶Re。

conjugates of the antibody and cytotoxic agent are prepared using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), Iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimide HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as benzylidene (tolyene)2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene). For example, ricin immunotoxins may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. (see WO 94/11026).

One of ordinary skill in the art will recognize that a wide variety of possible moieties may be conjugated to the resulting antibodies of the invention. (see, e.g., "coupling Vaccines", relations to Microbiology and Immunology, J.M. Cruse and R.E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

Conjugation can be accomplished by any chemical reaction that will bind the two molecules, as long as the antibody and other moieties retain their respective activities. The linkage may include a number of chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordination binding and complexation. However, preferred binding is covalent binding. Covalent bonding can be achieved by direct condensation of the side chains present or by incorporation of external bridging molecules. Many bivalent or multivalent linking agents may be used to couple protein molecules, such as the antibodies of the invention, to other molecules. For example, representative coupling agents may include organic compounds such as thioesters, carbodiimides, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzenes, and cyclohexanediamines. This list is not intended to exclude the various classes of coupling agents known in the art, but rather to exemplify more common coupling agents. (see Killen and Lindstrom, journal.Immun.133: 1335-2549 (1984); Jansen et al, Immunological Reviews 62:185-216 (1982); and Vitetta et al, Science 238:1098 (1987)).

Preferred linkers are described in the literature (see, e.g., Ramakrishan, S. et al, Cancer Res.44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester) and also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetyl hydrazide derivatives coupled to antibodies by means of oligopeptide linkers A particularly preferred linker comprises (i) EDC (1-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidooxycarbonyl- α -methyl- α - (2-pyridyl (pridyl) -dithio) -toluene (Pierce Chem. Co., Cat 21558G), (iii) SPDP (succinimido-6 [3- (2-pyridyldithio) propionamido ] hexanoate (Pierce-6 Co., Chem., Cat # 51G) (iv) thio-propionamido ] hexanoate (Pierce., Chem #9, S. 51, 2166-dithio-succinimido-6- (2-pyridyl) succinimide ester).

The linkers described above contain components that have different properties, thus resulting in conjugates with different physicochemical properties. For example, thio-NHS esters of alkyl carboxylates are more stable than thio-NHS esters of aromatic carboxylates. NHS-esters containing a linker are less soluble than thio-NHS esters. In addition, the linker SMPT contains sterically hindered disulfide bonds and can form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds, because disulfide bonds cleave in vitro, resulting in less available conjugates. Especially thio-NHS may enhance the stability of the carbodiimide coupling. When used with thio-NHS, carbodiimide coupling (such as EDC) forms esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as described in Epstein et al, Proc.Natl.Acad.Sci.USA, 82:3688 (1985), Hwang et al, Proc.Natl.Acad.Sci.USA, 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes are produced by a reverse evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to produce liposomes of the desired diameter. Fab' fragments of the antibodies of the invention can be conjugated to liposomes via a disulfide exchange reaction as described in Martin et al, J. biol. chem., 257: 286-.

Use of anti-CD 47x anti-MSLN antibodies

It will be understood that administration of the therapeutic entity in accordance with the present invention will be administered with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerance, and the like. A variety of suitable formulations can be found in all prescriptive sets known to pharmacists: remington's Pharmaceutical Sciences (15 th edition, Mack publishing company, Easton, Pa. (1975)), especially Chapter 87 by Blaug, Seymour, among others. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids (lipids), lipids (cationic or anionic) containing vesicles such as Lipofectin-cells, DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable in therapy and therapy according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerable with the route of administration. See also, Baldrick P. "Pharmaceutical excipient concentration. 32(2):210-8 (2000)," Wang W. "solubilization and expression vector of solid proteins pharmaceuticals." int. J. Pharm.203(1-2):1-60(2000), "Charman WN" Lipids, lipophillic drugs, and oral drug delivery-solvent concentrations. "J Pharm. Sci.89(8):967-78 (2000), P.E." composition for excipients for PDA J. Pharma. Sci. 238: 1998) and additional information on the Pharmaceutical carriers, see also excipients, well known to the skilled person for the present regulatory requirements, "Regulation for PDA J. Pharma. 52: 1998, and the Pharmaceutical formulations thereof.

Therapeutic formulations of the invention (which include antibodies of the invention) are useful for treating or alleviating symptoms associated with cancer, such as, but not limited to, leukemia, lymphoma, breast, colon, ovarian, bladder, prostate, glioma, lung and bronchial cancer, colorectal, pancreatic, esophageal, liver, bladder, kidney and renal pelvis, oral and pharyngeal cancer, uterine corpus, and/or melanoma. The invention also provides methods of treating or alleviating symptoms associated with cancer. The treatment regimen is carried out by identifying a subject, e.g., a human subject, having (or at risk of developing) cancer using standard methods.

Determining the effectiveness of the treatment in conjunction with any known method for diagnosing or treating a particular immune-related disorder. Alleviation of one or more symptoms of an immune-related disorder indicates that the antibody confers a clinical benefit.

Methods for screening for antibodies with the desired specificity include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and other immune-mediated techniques known in the art.

Antibodies directed against targets such as CD47, mesothelin, or combinations thereof (or fragments thereof) may be used in methods in the art that correlate with the localization and/or quantification of these targets, e.g., for measuring the levels of these targets in a suitable physiological sample, for diagnostic methods, for imaging proteins, etc.). In a given embodiment, antibodies, or derivatives, fragments, analogs or homologues thereof, containing an antibody-derived antigen-binding domain, specific for any of these targets are used as pharmaceutically active compounds (hereinafter "therapeutic agents").

The antibodies of the invention can be used to isolate specific targets using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation the antibodies of the invention (or fragments thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen¹²⁵I、¹³¹I、³⁵S or³H。

The present antibodies, including polyclonal, monoclonal, humanized and fully human antibodies, are useful as therapeutic agents, such agents will typically be used to treat or prevent diseases or pathologies associated with aberrant expression or activation of a given target in a subject.

A therapeutically effective amount of an antibody of the invention generally relates to the amount required to achieve a therapeutic target. As noted above, this can be a binding interaction between an antibody and its target antigen, which in some cases interferes with the function of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its particular antigen and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. A typical range of therapeutically effective doses of the antibodies or antibody fragments of the invention may be, but is not limited to, about 0.1 mg/kg body weight to about 50 mg/kg body weight. Typical dosing frequencies may range, for example, from twice daily to once weekly.

The antibodies or fragments thereof of the present invention may be administered in the form of pharmaceutical compositions for the treatment of a variety of diseases and disorders. Guidelines for the principles And considerations And selection Of components involved in the preparation Of such compositions are provided, for example, in Remington: the science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al, eds.) Mack pub.Co., Easton, Pa.:1995; Drug Absorption engineering: Concepts, Possibilites, Limitations, And Cold Trends, Harwood Academic Publishers, Langhorn, Pa., 1994; And Peptide And Protein Drug Delivery (Advance Inparteral Sciences, Vol. 4), 1991, M. Dekker, New York.

When antibody fragments are used, a minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules can be designed that retain the ability to bind to a target protein sequence based on the variable region sequence of the antibody. Such peptides may be chemically synthesized and/or produced by recombinant DNA techniques. (see, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-. The formulations may also contain more than one active compound necessary for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition may comprise an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.

The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions (macroemulsions).

The formulation to be used for in vivo administration must be sterile. This can be easily achieved by passing through sterile filtration membranes.

Sustained release formulations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

The antibodies according to the invention can be used as reagents for detecting the presence of a given target (or protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. The antibody is polyclonal, or more preferably, monoclonal. Using intact antibodies or fragments thereof (e.g. F)_abscFv, or F_(ab)2). The term "labeled" with respect to a probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of the primary antibody using a fluorescently labeled secondary antibody and end-labeling of the DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Thus, included in the use of the term "biological sample" are blood and fractions or components of blood,including serum, plasma or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assays (ELISAs), western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detecting genomic DNA of an analyte include Southern hybridization. Procedures for performing Immunoassays are described, for example, in "ELISA: the organism in Molecular Biology", Vol.42, J.R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E.Diamandis and T.Christopouus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P.Tijssen, Elsevier science publishers, Amsterdam, 1985. In addition, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte protein antibodies into a subject. For example, the antibody can be labeled with a radioactive label whose presence or location within the subject can be detected by standard imaging techniques.

Pharmaceutical composition

The antibodies of the invention (also referred to herein as "active compounds") and derivatives, fragments, analogs and homologs thereof can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are compatible with pharmaceutical administration. Suitable carriers are described in the latest versions of Remington's Pharmaceutical Sciences, which are standard references in the art and are incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds may also be incorporated into the compositions.

The pharmaceutical compositions of the present invention are formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be contained in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (in which water is soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM(BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria or fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, on the hydroxyl groupBenzoate, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions typically include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compounds can be incorporated with excipients and used in the form of tablets, lozenges or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and/or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients or compounds of similar properties: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, Primogel or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal use, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments (antigens), salves (salves), gels or creams as generally known in the art.

The compounds may also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is formulated with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Methods of preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

For ease of administration and consistency of dosage, it is especially advantageous to formulate oral or parenteral compositions in dosage unit form. Dosage unit form as used herein refers to physically discrete units (physically discrete units) suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as limitations inherent in the art of formulating such active compounds for use in the treatment of individuals.

The pharmaceutical composition may be included in a container, package, or dispenser along with instructions for administration.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Examples

Example 1: affinity measurement

An MSLN arm.The affinity of various anti-MSLN sequences (referred to herein as MSLN arms) was determined using the Bio-Layer interference (BLI) technique. The octatred 96 instrument and protein a biosensor (Pall, Basel, Switzerland) were used. Following the hydration and baseline steps, the biosensor was loaded with IgG at 1 μ g/mL in running buffer (PBS, NaCl, Tween 20, BSA, ProClin00) for 2 min. Control IgG was loaded on other biosensors for reference. The biosensor was then immersed in serial dilutions of human MSLN. According to the expectation of each candidateK _DThe MSLN concentration was adjusted. The biosensor was regenerated using 10 mM glycine pH 1.7. Affinity was measured using a 1:1 global fit model on a double reference curve. The results are shown below in table 5.

Table 5: affinity values for MSLN arms (n =2)

MSLN arm	K D [nM]
		O25	106 ± 9
O30	0.48 ± 0.03
		O32	529 ± 49
O35	22.7 ± 0.5
		O37	313 ± 74
O38	168 ± 23
		O41	19 ± 2

Example 2: cloning and expression of full-length cynomolgus mesothelin on the cell surface.

And (4) cloning.The sequence corresponding to the full-length protein cynomolgus mesothelin (cMSLN) was synthesized by Eurofins and provided in the pEX-K4 vector. DNA was prepared and digested with HindIII and EcoRI restriction enzymes. The insert was gel purified twice and cloned into pEAK8 EF1 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.). The constructs were verified by DNA sequencing.

And (4) expressing.The plasmid is then transfected into mammalian cells using a liposome-based transfection reagent such as Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA). The transfection step requires only a small amount of DNA and cells, typically 2. mu.g plasmid DNA and 2X10⁵Cells/well, and transfection was performed in 6-well plates. Although different mammalian cell lines can be used, in the examples given below, transformed human embryonic kidney monolayer epithelial cells (PEAK cells) are transfected. These cells stably express the EBNA-1 gene, further support the episomal replication process, are semi-adherent and can be cultured in standard conditioned cell culture chambers (5% CO2; 37 ℃ in DMEM supplemented with 10% fetal bovine serum)In nutrient medium). After 24 hours, the cells were placed under selection conditions by adding a medium containing 0.5-1. mu.g/mL puromycin, since the cells carrying the episomal vector were resistant to this antibiotic.

Cells were split in complete medium containing puromycin for generation of semi-stable cell lines expressing cMSLN. Cell lines are used for phage display selection and cell-based assays, such as fluorescence-related cell sorting (FACS).

Example 3: expression and purification of bispecific antibodies carrying lambda and kappa light chains

Simultaneous expression of one heavy chain and two light chains in the same cell can result in the assembly of three different antibodies. Simultaneous expression can be achieved in different ways such as transfection of multiple vectors expressing one of the strands for co-expression or by using vectors driving expression of multiple genes. The vector pNovi κ H λ was previously generated to allow co-expression of one heavy chain, one κ light chain and one λ light chain, as described in US 2012/0184716 and WO 2012/023053, each of which is incorporated herein by reference in its entirety. The expression of the three genes is driven by the human cytomegalovirus promoter (hCMV), and the vector also contains the glutamine synthetase Gene (GS) which enables the selection and establishment of stable cell lines. The VL gene of anti-hMSLN IgG λ or anti hCD47 IgG κ was cloned in the vector pNovi κ H λ for transient expression in mammalian cells. Peak cells were plated at 8X 10⁶The concentration of cells/flasks was expanded and split in T175 flasks in 45 ml of medium containing fetal bovine serum. 30 μ g of plasmid DNA was transfected into cells using Lipofectamine 2000 transfection reagent according to manufacturer's instructions. Antibody concentrations in serum-containing supernatants of transfected cells were measured at several time points during production using the Bio-Layer interference (BLI) technique. The OctetRED96 instrument and protein a biosensor were used for quantitation (Pall, Basel, Switzerland). Using 200 muL of supernatant for determining IgG concentration; the bioreactor was pre-conditioned and regenerated using 10 mM glycine ph1.7, and IgG calibrators (calibrators) diluted in conditioned (conditioned) PEAK cell culture medium were prepared for standard curve generation. Y-standard curve weighted using dose response 5PLThe line equation and initial slope are combined with the rate equation to determine concentration. Supernatants were harvested 7-10 days post transfection and clarified by centrifugation at 1300 g for 10 min, depending on antibody concentration. The purification process consists of three affinity steps. First, CaptureSelect ­ IgG-CH1 affinity matrix (ThermoFisher Scientific, Waltham, MA) was washed with PBS and then added to the clarified supernatant. After overnight incubation at +4 ℃, the supernatant was centrifuged at 1000 g for 10 min, the flow through was stored (flow through) and the resin was washed twice with PBS. Subsequently, the resin was transferred to a spin column and a solution containing 50 mM glycine pH 2.7 was used for elution. Several elution fractions were generated, pooled and desalted with 25mM histidine/125 mM NaCl pH6.0 buffer using 50kDa Amicon @ Ultra Centrifugal filter units (Merck KGaA, Darmstadt, Germany). The final product containing total human IgG from the supernatant was quantified using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) and incubated at RT and 20 rpm with an appropriate volume of CaptureSelect LC-kappa (Hu) affinity matrix (Thermo Fisher scientific, Waltham, MA) for 30 minutes. Incubation, resin recovery, elution and desalting steps were performed as described previously. The final affinity purification step was performed using a CaptureSelect LC-lambda (hu) affinity matrix (Thermo FisherScientific, Waltham, MA) as applied to the same method for the previous two purification steps. The final product was quantified using Nanodrop. Purified bispecific antibodies were analyzed by electrophoresis under denaturing and reducing conditions. Agilent 2100 Bioanalyzer and Protein 80 kits (Agilent Technologies, Santa Clara, Calif., USA) were used as described by the manufacturer. mu.L of the purified sample was mixed with sample buffer supplemented with dithiothreitol (DTT; Sigma Aldrich, St. Louis, MO). The sample was heated at 95 ℃ for 5 minutes and then loaded onto the chip. Aliquots from the first purification step (containing bispecific antibody and two monospecific mabs) and the final product were loaded on an isoelectric focusing (IEF) gel to assess the purity of the final purified bispecific antibody (absence of mAb contamination). Aggregate levels were determined by SEC-HPLC. Finally, the binding of the bispecific antibody on both targets was evaluated using OctetRED 96. Briefly, biotinylated targets (hMSLN, hCD47 and unrelated targets)) Loaded on a streptavidin biosensor. The biosensor is then immersed in a solution containing the bispecific antibody and binding is monitored in real time. All samples were tested for endotoxin contamination using the Limulus Amebocyte Lysate test (LAL; Charles River Laboratories, Wilmington, Mass.).

Example 4: cloning, expression and purification of human mesothelin

And (4) cloning.The sequence corresponding to the extracellular domain (amino acids 296 to 580) of human mesothelin (hMSLN), followed by an Avitag (Avidity, Denver CO) and a C-terminal hexahistidine tag was synthesized by Eurofins and provided in a pEX-K vector. The tags allow single site biotinylation of the protein and purification by IMAC (immobilized metal ion affinity chromatography), respectively. DNA was prepared and digested with HindIII and EcoRI restriction enzymes. The insert was gel purified twice and cloned into pEAK8 EF1 mammalian expression vector (Edge Biosystems, Gaithersburg, Md.). The construct was verified by DNA sequencing.

And (4) expressing.The plasmid is then transfected into mammalian cells using a liposome-based transfection reagent such as Lipofectamine 2000 (ThermoFisher Scientific, Waltham, MA). The transfection step requires only a small amount of DNA and cells, typically 2X10⁵Individual cells and 2 μ g plasmid DNA per well, and transfection was performed in 6-well plates. Although different mammalian cell lines can be used, in the examples given below, transformed human embryonic kidney monolayer epithelial cells (PEAK cells) are transfected. These cells stably express the EBNA-1 gene, further supporting the episomal replication process, are semi-adherent and can be grown in standard conditioned cell culture chambers (5% CO2; 37 ℃ in DMEM medium supplemented with 10% fetal bovine serum). After 24 hours, the cells were placed under selection conditions by adding a medium containing 0.5-1. mu.g/mL puromycin, since the cells carrying the episomal vector were resistant to this antibiotic.

Two to three weeks after transfection, cells were used to inoculate a disposable CELLine bioreactor for the production step. For in vivo production of biotinylated proteins, 50 μ M biotin was added to the medium. CELLine is a two-compartment bioreactor that can be used within standard cell culture chambers. The smaller compartment (15 mL) contained cells in serum-free medium and was separated from the larger (one liter) compartment containing complete medium by a semipermeable membrane with a cut-off size of 10 kDa (Bruce et al 2002, McDonald et al 2005). This system allows diffusion of nutrients, gazes and metabolic waste products while retaining cells and secreted proteins in smaller compartments. Culture was maintained for 7-10 days before harvesting the supernatant. Since the medium contains serum, the cells maintain good viability and several production runs can be performed using the same cells and vessels.

And (5) purifying.After harvesting, the supernatant recovered from the cell compartment of the CELLine bioreactor contains concentrated recombinant protein and low levels of contaminants because they cannot cross the 10 kDa membrane separating the two chambers of the reactor this increased ratio of recombinant protein to contaminants greatly enhances the purification efficiency using IMAC the supernatant is clarified by centrifugation and filtration through a 0.22 μm membrane followed by addition of 100 mM imidazole and loading onto a Ni-NTA affinity chromatography resin (Qiagen) the binding of contaminants to the resin is minimized by the relatively high concentration of imidazole after washing the column, the proteins are eluted on an Ä KTAPrime chromatography system (GE Healthcare, Little Chalfont, UK) using a 30 mL imidazole gradient (20-400 mM imidazole) at a flow rate of 2 mL/min after washing the column, their content in the recombinant protein can be determined by SDS-PAGE or biotinylation analysis.

Example 5: immobilized VH candidate recombination (reformat) to IgG and transient expression in mammalian cells

After screening, scFv candidates against hMSLN or hCD47 were recombined into IgG and transiently transfected into PEAK cells. The VL sequence of the selected scFv was amplified with specific oligonucleotides and cloned into an expression vector containing universal heavy and light chain constant regions. Construction was verified by sequencing. Mammalian Peak cells were plated at 3 × 10 in 25 mL fetal bovine serum-containing medium in T75 flasks⁶Concentration of individual cells/flask at 37 ℃ and 5% CO₂The cells were grown in a humidified incubator. One day after cell division, the expression vector was transfected using Lipofectamine 2000 transfection reagent (Thermo Fisher scientific, Waltham, Mass.) according to the manufacturer's instructions. Antibody concentrations in serum-containing supernatants of transfected cells were measured at several time points during production using the Bio-Layer interference (BLI) technique. The OctetRED96 instrument and protein a biosensor were used for quantitation (Pall, Basel, Switzerland). Using 200 muL of supernatant for determining IgG concentration; the bioreactor was pre-conditioned and regenerated using 10 mM glycine pH1.7, and IgG calibrators (calibretors) diluted in conditioned (conditioned) Peak cell culture media were prepared for standard curve generation. Concentrations were determined using a dose-response 5PL unweighted standard curve equation and an initial slope binding rate equation. After 6-7 days of culture, supernatants were harvested for IgG purification on FcXL affinity chromatography resin (Thermo FisherScientific, Waltham, MA) according to the manufacturer's instructions. Briefly, supernatants from transfected cells were incubated with the resin overnight at +4 ℃. The sample was then centrifuged and the resin was transferred to a column filter for elution. The eluted IgG fractions were then desalted against PBS and the IgG content quantified by absorbance at 280 nm. Purity and IgG integrity were verified by electrophoresis.

Example 6: binding of MSLN monoclonal antibody and MSLN/CD47 bispecific antibody to human and cynomolgus monkey mesothelin

The ability of MSLN monoclonal antibodies and MSLN/CD47 bispecific antibodies to bind cell surface expressed human and cynomolgus mesothelin was tested by flow cytometry. CHO cells stably expressing human MSLN (CHO-humSLN cells) or cynomolgus MSLN (CHO-humSLN cells) were used for this purpose, since the anti-human CD47 antibody arm of the biAb does not recognize hamster mesothelin orthologs. Briefly, increasing concentrations of MSLN Mab and biAb were incubated with CHO-humLN cells or CHO-cyMSLN cells at 4 ℃ for 30 minutes. After two washes, bound antibody was detected using PE-conjugated anti-human Fc secondary antibody (Southern Biotech # 9042-09). Untransfected CHO cells were used as a control. Figure 1 shows strong binding of MSLN monoclonal and bispecific antibodies to mesothelin expressed on the surface of mesothelin-transfected CHO cells. All MSLN antibodies showed a high level of species cross-reactivity, as the MFI/antibody concentration curves obtained with CHO cells expressing human and cynomolgus MSLN appeared very similar.

Example 7: ADCP induced by bispecific antibodies targeting MSLN and CD 47.

The ability to dual target the CD47/MSLN κ λ body to co-link CD47 and MSLN on the cell surface allows neutralization of the MSLN-dependent CD47-SIRPa interaction. This in turn translates into efficient and selective cancer cell killing mediated by the CD47/MSLN κ λ body, as demonstrated in the ADCP experiments described in this example. Three tumor cell lines expressing different levels of CD47 and MSLN were tested for ADCP: NCI-N87, HPAC, and Caov-3. The cell surface expression levels of CD47 and mesothelin of NCI-N87 cells were 43,000 and 27,000, respectively. The cell surface expression levels of CD47 and mesothelin for HPAC cells were 105,000 and 13,000, respectively. The cell surface expression levels of CD47 and mesothelin for Caov-3 cells were 220,000 and 38,000, respectively. ADCP experiments were performed with human macrophages differentiated from peripheral blood mononuclear cells. Two different assay formats were used to assess phagocytosis. In the experiment shown in FIG. 2, macrophages were incubated with CFSE-labeled target cells (effector: target ratio 3: 1) at 37 ℃ for 2.5 hours in the presence of increasing concentrations of antibody. At the end of the incubation period, biotinylated anti-human CD1 antibody and Strep-Cy5 were added to label macrophages. The cells were then washed and subjected to FACS analysis. Phagocytosis was confirmed by double positive events. In the experiment shown in fig. 3, macrophages adhering to the well of a microporous plate were incubated with calcein AM-labeled target cells (effector: target ratio 1:1) in the presence of increasing concentrations of antibody for 2.5 hours at 37 ℃. At the end of the incubation period, the supernatant was replaced by complete medium and the microplates were imaged with a CellInsight CX5 high content screening platform. 1500 macrophages were harvested and analyzed per well. Phagocytosis was demonstrated as a double positive event and the phagocytosis index was calculated by software.

The dose-response experiment in figure 2 demonstrates that CD47/MSLN BsAb phagocytose NCI-N87 and HPAC cells in an MSLN-dependent manner, given the much less efficient use of a monovalent antibody to CD47 (i.e., the κ λ body lacking the anti-MSLN arm). FIG. 2 also shows that CD47/MSLN BsAb induced ADCP more efficiently than the benchmark antibody, the high affinity anti-human CD47 monoclonal antibody B6H12-huIgG1 or the monoclonal MSLN antibody amateximab (KEGG ID: D09767, PubChem SID: 124490507). FIG. 3 shows a comparison of ADCP with the CD47/MSLN κ λ body with the corresponding anti-CD 47 and anti-MSLN monovalent antibodies and anti-MSLN mAbs. The CD47/MSLN κ λ bodies induced significantly higher levels of NCI-N87 and Caov-3 target cell phagocytosis, confirming that dual target ligation and MSLN-mediated CD47 blockade are critical for in vitro efficacy.

Example 8: ADCC induced by bispecific antibodies targeting MSLN and CD 47.

Antibody-dependent cell-mediated cytotoxicity (ADCC) of four CD47/MSLN κ λ bodies (K2O25, K2O35, K2O38 and K2O41) was evaluated using a Cr 51-releasing cell-based assay. Three tumor cell lines expressing different levels of CD47 and MSLN were tested for ADCC: NCI-N87, NCI-H226, and HepG 2-MSLN. The cell surface expression levels of CD47 and mesothelin of NCI-N87 cells were 43,000 and 27,000, respectively. The cell surface expression levels of CD47 and mesothelin of NCI-H226 cells were 200,000 and 250,000, respectively. The cell surface expression levels of CD47 and mesothelin of HepG2-MSLN cells (obtained by stable transfection of human mesothelin in the human hepatoma cell line HepG 2) were 22,000 and 120,000, respectively. ADCC experiments were performed with whole human PBMC and Cr 51-loaded MSLN-positive target cell line as effector cells. Briefly, target cells were loaded with Cr51 for 1 hour at 37 ℃. After washing, cells were conditioned with CD47/MSLN κ λ bodies or the MSLN reference mAb Amatoximab for 30 min at 37 ℃. Then 5,000 Cr 51-loaded target cells were mixed with 250,000 IL-2 activated PBMC effector cells to obtain a final 50:1 ratio between effector and target cells (E: T ratio =50) and incubated at 37 ℃ for 4 h. After a short centrifugation (10 min at 1500 rpm), the cell-free supernatant was counted in a gamma-counter. The negative control (spontaneous Cr51 release) consisted of Cr 51-loaded target cells incubated with medium in the absence of effector cells. The total lysis control consisted of Cr 51-loaded target cells incubated with a cell lysis solution (Triton X-100). The non-specific lysis control (baseline) consisted of Cr 51-loaded target cells incubated with effector cells without addition of any antibody. ADCC reactions were performed in triplicate. Ab specific ADCC percentage was calculated using the formula: % ADCC = ((sample cpm-nonspecific lysis control cpm)/(total lysis control cpm-negative control cpm)) x 100%. The experiment shown in FIG. 4 compares the effects of four CD47/MSLN κ bodies and the MSLN reference mAb amauximab. All CD47/MSLN κ λ tested exhibited approximately similar ADCC potency with the three cell lines. In all cases, ADCC induced by CD47/MSLN κ bodies was significantly higher than that of amauximab.

Example 9: in vivo anti-tumor Activity of bispecific antibodies

Five CD47/MSLN κ λ bodies (biAb025, biAb030, biAb035, biAb038, and biAb041) were evaluated for anti-tumor activity in xenograft models. In the experiment shown in FIG. 5, 3 is addedx 10⁶Individual HepG2-MSLN cells were implanted subcutaneously into NOD/SCID mice and allowed to grow for 15 days. Subsequently, the mice were randomly divided into 6 groups (7 mice per group) and antibody treatment was started. Antibodies were injected i.v. once a week until the end of the experiment (d 55). In the experiment shown in FIG. 6A, 3 was runx 10⁶Individual OVCAR3 cells were implanted subcutaneously in NOD/SCID mice. The following day, mice were randomly divided into 2 groups (7 mice per group) and antibody treatment was started. Antibodies were injected i.v. once a week until d 56. In the experiment shown in FIG. 6B, 3 was runx 10⁶Individual CAOV3 cells were implanted subcutaneously in NOD/SCID mice. The following day, mice were randomly divided into 2 groups (6 or 7 mice per group) and antibody treatment was initiated. Antibodies were injected i.v. once a week until d 18. All antibodies were administered at 60mg/kg per injection. Tumor volume was measured 2 to 3 times per week and calculated using the formula: ((length x width 2)/2). The embodiment shown in FIG. 5The assay compares the effect of CD47/MSLN kappa lambda antibody with the reference monoclonal antibodies CD47 mAb B6H12-hIgGl and MSLN mAb amateximab. For statistical analysis at the endpoint (fig. 5A), one-way ANOVA was performed using GraphPad Prism, followed by multiple comparison test (Tukey's multiple comparison). p-value: p<0.05，**p<0.01; ns, not significant. The percentage of Tumor Growth Inhibition (TGI) compared to the isotype control group was also determined using the following formula (fig. 5 b): % TGI = {1- [ (Tt-TO)/(Vt-VO)]X 100; wherein Tt = median tumor volume treated at time t; TO = median tumor volume treated at time 0; vt = median tumor volume of control at time t, and VO = median tumor volume of control at time = O. As shown in figure 5, treatment with five CD47/MSLN κ λ -bodies and amateximab, but not CD47 mAb B6H12-hlgG1, significantly reduced tumor growth compared to the hlgG1 control. Furthermore, four of the five CD47/MSLN κ λ -bodies tested (all except biAb 030) all had superior anti-tumor efficacy to amateuximab, with three biabs exhibiting TGI>90% (biAb025, biAb038 and biAb 041). The experiment shown in FIG. 6 shows that the body of biabO38 CD47/MSLN κ prevents tumor growth.

Other embodiments

While the present invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (30)

1. An isolated bispecific antibody comprising a first arm comprising a first amino acid sequence that binds CD47 and a second arm comprising a second amino acid sequence that binds Mesothelin (MSLN), wherein the bispecific antibody inhibits the interaction between CD47 and signal-regulating protein α (SIRP α).

2. The isolated bispecific antibody of claim 1, wherein the bispecific antibody inhibits the interaction between human CD47 and human SIRP α.

3. The isolated bispecific antibody of claim 1, wherein the first arm comprises: the variable heavy chain complementarity determining region 1(CDRH1) amino acid sequence of SEQ ID NO:225, the variable heavy chain complementarity determining region 2(CDRH2) amino acid sequence of SEQ ID NO:226, the variable heavy chain complementarity determining region 3(CDRH3) amino acid sequence of SEQ ID NO:227, the variable light chain complementarity determining region 1(CDRL1) amino acid sequence selected from SEQ ID NOS 228-241 and 262-272, the variable light chain complementarity determining region 2(CDRL2) amino acid sequence selected from SEQ ID NOS-242-245 and 273-280, and the variable light chain complementarity determining region 3(CDRL3) amino acid sequence selected from SEQ ID NOS-246-261 and 281.

4. The isolated bispecific antibody of claim 1, wherein the first arm comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain amino acid sequence selected from SEQ ID NO 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204 and 206.

5. The isolated bispecific antibody of claim 1, wherein the first arm comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114 and a variable light chain comprising the amino acid sequence of SEQ ID NO 168.

6. The isolated bispecific antibody of claim 1, wherein the first arm comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO. 2 and a light chain comprising the amino acid sequence of SEQ ID NO. 56.

7. The isolated bispecific antibody of claim 1, wherein the second arm comprises: the amino acid sequence of CDRH1 of SEQ ID NO:225, the amino acid sequence of CDRH2 of SEQ ID NO:226, the amino acid sequence of CDRH3 of SEQ ID NO:227, the amino acid sequence of CDRL1 selected from SEQ ID NO: 282-containing 287, the amino acid sequence of CDRL2 selected from SEQ ID NO: 288-containing 293, and the amino acid sequence of CDRL3 selected from SEQ ID NO: 294-containing 300.

8. The isolated bispecific antibody of claim 1, wherein the second arm comprises the CDRH1 amino acid sequence of SEQ ID NO:225, the CDRH2 amino acid sequence of SEQ ID NO:226, the CDRH3 amino acid sequence of SEQ ID NO:227, and a combination of the CDRL1 amino acid sequence, the CDRL2 amino acid sequence, and the CDRL3 amino acid sequence selected from the group consisting of SEQ ID NO:

(a) CDRL1 comprising the amino acid sequence of SEQ ID NO:282, CDRL2 comprising the amino acid sequence of SEQ ID NO:288 and CDRL3 comprising the amino acid sequence of SEQ ID NO: 294;

(b) CDRL1 comprising the amino acid sequence of SEQ ID NO:283, CDRL2 comprising the amino acid sequence of SEQ ID NO:289, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 295;

(c) CDRL1 comprising the amino acid sequence of SEQ ID NO:284, CDRL2 comprising the amino acid sequence of SEQ ID NO:290 and CDRL3 comprising the amino acid sequence of SEQ ID NO: 296;

(d) CDRL1 comprising the amino acid sequence of SEQ ID NO:285, CDRL2 comprising the amino acid sequence of SEQ ID NO:291 and CDRL3 comprising the amino acid sequence of SEQ ID NO: 297;

(e) CDRL1 comprising the amino acid sequence of SEQ ID NO. 286, CDRL2 comprising the amino acid sequence of SEQ ID NO. 292 and CDRL3 comprising the amino acid sequence of SEQ ID NO. 298;

(f) CDRL1 comprising the amino acid sequence of SEQ ID NO 287, CDRL2 comprising the amino acid sequence of SEQ ID NO 293 and CDRL3 comprising the amino acid sequence of SEQ ID NO 299; and

(g) CDRL1 comprising the amino acid sequence of SEQ ID NO. 282, CDRL2 comprising the amino acid sequence of SEQ ID NO. 288 and CDRL3 comprising the amino acid sequence of SEQ ID NO. 300.

9. The isolated bispecific antibody of claim 1, wherein the second arm comprises: the variable heavy chain amino acid sequence of SEQ ID NO 114 and the variable light chain comprising an amino acid sequence selected from SEQ ID NO 212, 214, 216, 218, 220, 222 and 224.

10. The isolated bispecific antibody of claim 1, wherein the second arm comprises the heavy chain amino acid sequence of SEQ ID No. 2 and a light chain amino acid sequence selected from the group consisting of SEQ ID nos. 98, 100, 102, 104, 106, 108, and 110.

11. The isolated bispecific antibody of claim 1, wherein the isolated bispecific antibody comprises: a heavy chain comprising CDRH1 comprising the amino acid sequence comprising SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a kappa light chain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242 and CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising a CDRL1 amino acid sequence selected from the group consisting of SEQ ID NO:282-287, a CDRL2 amino acid sequence selected from the group consisting of SEQ ID NO:288-293 and a CDRL3 amino acid sequence selected from the group consisting of SEQ ID NO: 294-300.

12. The isolated bispecific antibody of claim 1, wherein the isolated bispecific antibody comprises: a heavy chain comprising CDRH1 comprising the amino acid sequence of SEQ ID NO:225, CDRH2 comprising the amino acid sequence of SEQ ID NO:226, CDRH3 comprising the amino acid sequence of SEQ ID NO:227, a CDRL1 comprising the amino acid sequence of SEQ ID NO:240, CDRL2 comprising the amino acid sequence of SEQ ID NO:242 and a kappa light chain comprising CDRL3 comprising the amino acid sequence of SEQ ID NO:254, and a lambda light chain comprising a combination of a CDRL1 amino acid sequence, a CDRL2 amino acid sequence, and a CDRL3 amino acid sequence selected from the group consisting of SEQ ID NO:

(a) CDRL1 comprising the amino acid sequence of SEQ ID NO:282, CDRL2 comprising the amino acid sequence of SEQ ID NO:288 and CDRL3 comprising the amino acid sequence of SEQ ID NO: 294;

(b) CDRL1 comprising the amino acid sequence of SEQ ID NO:283, CDRL2 comprising the amino acid sequence of SEQ ID NO:289, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 295;

(c) CDRL1 comprising the amino acid sequence of SEQ ID NO:284, CDRL2 comprising the amino acid sequence of SEQ ID NO:290 and CDRL3 comprising the amino acid sequence of SEQ ID NO: 296;

(d) CDRL1 comprising the amino acid sequence of SEQ ID NO:285, CDRL2 comprising the amino acid sequence of SEQ ID NO:291 and CDRL3 comprising the amino acid sequence of SEQ ID NO: 297;

(e) CDRL1 comprising the amino acid sequence of SEQ ID NO. 286, CDRL2 comprising the amino acid sequence of SEQ ID NO. 292 and CDRL3 comprising the amino acid sequence of SEQ ID NO. 298;

(f) CDRL1 comprising the amino acid sequence of SEQ ID NO 287, CDRL2 comprising the amino acid sequence of SEQ ID NO 293 and CDRL3 comprising the amino acid sequence of SEQ ID NO 299; and

(g) CDRL1 comprising the amino acid sequence of SEQ ID NO. 282, CDRL2 comprising the amino acid sequence of SEQ ID NO. 288 and CDRL3 comprising the amino acid sequence of SEQ ID NO. 300.

13. The isolated bispecific antibody of claim 1, wherein the isolated bispecific antibody comprises: a variable heavy chain comprising the amino acid sequence of SEQ ID NO 114, a kappa variable light chain comprising the amino acid sequence of SEQ ID NO 168, and a lambda variable light chain comprising an amino acid sequence selected from SEQ ID NO 212, 214, 216, 218, 220, 222, and 224.

14. The isolated bispecific antibody of claim 1, wherein the isolated bispecific antibody comprises: a variable heavy chain comprising the amino acid sequence of SEQ id No. 114, and a combination of a kappa variable light chain and a lambda light chain selected from the group consisting of:

(a) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO:168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO: 212;

(b) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO:168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO: 214;

(c) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO:168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO: 216;

(d) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO. 168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO. 218;

(e) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO:168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO: 220;

(f) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO. 168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO. 222; and

(g) a kappa variable light chain comprising the amino acid sequence of SEQ ID NO:168 and a lambda variable light chain comprising the amino acid sequence of SEQ ID NO: 224.

15. The isolated bispecific antibody of claim 1, wherein the isolated bispecific antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID No. 2, a kappa light chain comprising the amino acid sequence of SEQ ID No. 56, and a lambda light chain comprising an amino acid sequence selected from SEQ ID Nos. 98, 100, 102, 104, 106, 108 and 110.

16. The isolated bispecific antibody of claim 1, wherein the isolated bispecific antibody comprises: a heavy chain comprising the amino acid sequence of SEQ id No. 2, and a combination of a kappa light chain and a lambda light chain selected from the group consisting of:

(a) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 98;

(b) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 100;

(c) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 102;

(d) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 104;

(e) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 106;

(f) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 108; and

(g) a kappa light chain comprising the amino acid sequence of SEQ ID NO. 56 and a lambda light chain comprising the amino acid sequence of SEQ ID NO. 110.

17. The isolated bispecific antibody of any one of claims 1 to 16, wherein the bispecific antibody comprises two copies of a single heavy chain polypeptide and a first light chain and a second light chain, wherein the first and second light chains are different.

18. The isolated bispecific antibody of claim 17, wherein at least a portion of the first light chain is of the kappa type and at least a portion of the second light chain is of the lambda type.

19. The isolated bispecific antibody of claim 18, wherein the first light chain comprises at least a kappa constant region.

20. The isolated bispecific antibody of claim 19, wherein the first light chain further comprises a kappa variable region.

21. The isolated bispecific antibody of claim 19, wherein the first light chain further comprises a λ variable region.

22. The isolated bispecific antibody of claim 18, wherein the second light chain comprises at least a lambda constant region.

23. The isolated bispecific antibody of claim 22, wherein the second light chain further comprises a lambda variable region.

24. The isolated bispecific antibody of claim 22, wherein the second light chain further comprises a kappa variable region.

25. The isolated bispecific antibody of claim 18, wherein the first light chain comprises a kappa constant region and a kappa variable region, and wherein the second light chain comprises a lambda constant region and a lambda variable region.

26. The isolated bispecific antibody of any one of claims 1 to 25, wherein the constant and variable framework region sequences are human.

27. Use of the isolated bispecific antibody of any one of claims 1-26 for treating, preventing or delaying the progression of a pathology associated with aberrant CD47 expression or activity or associated with aberrant CD47-SIRP α expression or activity.

28. The use of claim 27, wherein the pathology is cancer.

29. The use of claim 28, wherein the cancer is a solid tumor.

30. The use of claim 29, wherein the solid tumor is or is derived from breast cancer, ovarian cancer, head and neck cancer, bladder cancer, melanoma, mesothelioma, colorectal cancer, cholangiocarcinoma, pancreatic cancer, lung cancer, leiomyoma, leiomyosarcoma, renal cancer, glioma, glioblastoma, endometrial cancer, esophageal cancer, biliary tract gastric cancer, prostate cancer, or a combination thereof.

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