Combination of a bispecific fusion protein and an anti-Her2 antibody for tumor treatment

Abstract

The present invention relates to the use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating a tumor in a subject in need thereof, and further to a pharmaceutical composition comprising an effective amount of said immune checkpoint inhibitor and an effective amount of said Her2 inhibitor, and optionally a pharmaceutically acceptable excipient, and the use of the pharmaceutical composition in the preparation of a medicament for treating a tumor in a subject in need thereof.

Description

Technical Field

[0001] The present invention relates to the use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumors in a subject in need of an immune checkpoint inhibitor.

Background Art

[0002] The immune system plays an important role in the antitumor effect of anti-Her2 antibodies such as the ADCC effect of anti-Her2 antibodies and is related to T cell function. The higher the TIL value of a patient, the better the response to anti-Her2 antibodies. Conversely, patients who do not achieve pathological complete remission (pCR) have elevated Treg levels and exhibit an immunosuppressive state.

[0003] Animal experiments have also shown that trastuzumab increases the secretion of IFN-γ and thereby induces the expression of PDL1. This pathway may also be one of the mechanisms of trastuzumab resistance.

[0004] Preclinical studies have confirmed that the therapeutic effect of Her2 targeting depends on the adaptive immune response of the body. Therefore, by combining with tumor immunity-related targets (such as PD1 / PDL1, CTLA4, 4-1BB, etc.), the antitumor effect of the drug can be synergistically enhanced.

Disclosure of the Invention

Problems to be Solved by the Invention

[0005] The present disclosure provides the use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumors in a subject in need of an immune checkpoint inhibitor that can specifically bind to PD-L1 and CTLA4, a pharmaceutical composition comprising an effective amount of the immune checkpoint inhibitor and an effective amount of the Her2 inhibitor, and optionally a pharmaceutically acceptable excipient, and the use of the pharmaceutical composition in the preparation of a medicament for treating tumors in a subject in need thereof.

Means for Solving the Problem

[0006] In one form, the present disclosure provides the use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumors in a subject in need thereof, wherein the immune checkpoint inhibitor can specifically bind to PD-L1 and CTLA4.

[0007] In some embodiments, the immune checkpoint inhibitor is a bispecific antibody or an antigen-binding fragment thereof.

[0008] In some embodiments, the immune checkpoint inhibitor is a bispecific antibody, and the bispecific antibody is a fully human antibody.

[0009] In some embodiments, the immune checkpoint inhibitor is an antigen-binding fragment, and the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0010] In some embodiments, the immune checkpoint inhibitor is a dimer, the dimer is formed by two polypeptide chains, each of the two polypeptide chains includes an antibody Fc subunit, the dimer includes two or more immunoglobulin single variable domains (ISVDs), at least one of the ISVDs is specific for PD-L1, and at least one of the ISVDs is specific for CTLA4.

[0011] In some embodiments, at least one of the two polypeptide chains includes both an ISVD specific for PD-L1 and an ISVD specific for CTLA4.

[0012] In some embodiments, each of the two polypeptide chains includes both an ISVD specific for PD-L1 and an ISVD specific for CTLA4.

[0013] In some embodiments, for one or both of the two polypeptide chains, the ISVD specific for PD-L1 is fused to the ISVD specific for CTLA4, optionally via a linker.

[0014] In some embodiments, for one or both of the two polypeptide chains: the ISVD specific for PD-L1 is fused, optionally via a linker, to the ISVD specific for CTLA4; and the ISVD specific for CTLA4 is fused, optionally via a linker, to the antibody Fc subunit.

[0015] In some embodiments, for one or both of the two polypeptide chains: the C-terminus of the ISVD specific for PD-L1 is fused, optionally via a linker, to the N-terminus of the ISVD specific for CTLA4; and the C-terminus of the ISVD specific for CTLA4 is fused, optionally via a linker, to the N-terminus of the antibody Fc subunit.

[0016] In some embodiments, for one or both of the two polypeptide chains: the ISVD specific for PD-L1 is fused, optionally via a linker, to the ISVD specific for CTLA4; and the ISVD specific for PD-L1 is fused, optionally via a linker, to the antibody Fc subunit.

[0017] In some embodiments, for one or both of the two polypeptide chains: the C-terminus of the ISVD specific for CTLA4 is fused, optionally via a linker, to the N-terminus of the ISVD specific for PD-L1; and the C-terminus of the ISVD specific for PD-L1 is fused, optionally via a linker, to the N-terminus of the antibody Fc subunit.

[0018] In some embodiments, the antibody Fc subunit is derived from an IgG Fc subunit.

[0019] In some embodiments, the IgG is human IgG1.

[0020] In some embodiments, the antibody Fc subunit comprises an amino acid sequence set forth in any one of SEQ ID NOs: 35, 38, and 39.

[0021] In some embodiments, the ISVD specific for PD-L1 is capable of binding to the N-terminal IgV domain of human PD-L1.

[0022] In some embodiments, the ISVD specific for PD-L1 can bind to residues I54, Y56, E58, Q66, and / or R113 of the human PD-L1 N-terminal IgV domain, and the human PD-L1 N-terminal IgV domain comprises the amino acid sequence set forth in SEQ ID NO:64.

[0023] In some embodiments, the ISVD specific for PD-L1 may further bind to residues D61, N63, V68, M115, S117, Y123 and / or R125 of the human PD-L1 N-terminal IgV domain, wherein the human PD-L1 N-terminal IgV domain comprises the amino acid sequence set forth in SEQ ID NO:64.

[0024] In some embodiments, the ISVD specific for PD-L1 is capable of binding to a conformational epitope in the human PD-L1 N-terminal IgV domain, wherein the conformational epitope comprises residues I54, Y56, E58, Q66, and R113 of the human PD-L1 N-terminal IgV domain, and wherein the human PD-L1 N-terminal IgV domain comprises the amino acid sequence set forth in SEQ ID NO:64.

[0025] In some embodiments, the ISVD specific for PD-L1 is capable of binding to a conformational epitope in the human PD-L1 N-terminal IgV domain, wherein the conformational epitope comprises residues I54, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123, and R125 of the human PD-L1 N-terminal IgV domain, and the human PD-L1 N-terminal IgV domain comprises the amino acid sequence set forth in SEQ ID NO:64.

[0026] In some embodiments, the ISVD specific for PD-L1 can block the binding of PD-L1 to PD1.

[0027] In some embodiments, the ISVD specific for PD-L1 can block the binding of PD-L1 to CD80.

[0028] In some embodiments, the ISVD specific for PD-L1 competes for the binding of the reference anti-PD-L1 antibody to PD-L1, wherein the reference anti-PD-L1 antibody comprises a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0029] In some embodiments, the reference anti-PD-L1 antibody comprises a heavy chain CDR3 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 9.

[0030] In some embodiments, the reference anti-PD-L1 antibody comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0031] In some embodiments, the reference anti-PD-L1 antibody comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 and 7.

[0032] In some embodiments, the reference anti-PD-L1 antibody comprises a heavy chain CDR2 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 4, 8, and 11.

[0033] In some embodiments, the reference anti-PD-L1 antibody is an ISVD specific for PD-L1.

[0034] In some embodiments, the reference anti-PD-L1 antibody comprises a heavy chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15.

[0035] In some embodiments, the reference anti-PD-L1 antibody comprises a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0036] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[0037] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain CDR3 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 9.

[0038] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 2.

[0039] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain CDR1 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 and 7.

[0040] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain CDR2 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 4, 8, and 11.

[0041] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15.

[0042] In some embodiments, the ISVD specific for PD-L1 comprises a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO: 6.

[0043] In some embodiments, the ISVD specific for CTLA4 can specifically bind to human CTLA4.

[0044] In some embodiments, the ISVD specific for CTLA4 can block the binding of CTLA4 to CD80.

[0045] In some embodiments, the ISVD specific for CTLA4 can block the binding of CTLA4 to CD86.

[0046] In some embodiments, the ISVD specific for CTLA4 cross-competes with respect to the binding of a reference anti-CTLA4 antibody to CTLA4, and the reference anti-CTLA4 antibody comprises a heavy-chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 19.

[0047] In some embodiments, the reference anti-CTLA4 antibody comprises a heavy-chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 17.

[0048] In some embodiments, the reference anti-CTLA4 antibody comprises a heavy-chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 16.

[0049] In some embodiments, the reference anti-CTLA4 antibody comprises a heavy-chain CDR2 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 21, and 23.

[0050] In some embodiments, the reference anti-CTLA4 antibody is an ISVD specific for CTLA4.

[0051] In some embodiments, the reference anti-CTLA4 antibody comprises a heavy-chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 20, 22, and 24-32.

[0052] In some embodiments, the reference anti-CTLA4 antibody comprises a heavy-chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO: 20.

[0053] In some embodiments, the long-lived state is evaluated by detecting the survival time.

[0054] In some embodiments, the cognitive state is evaluated by an open-field experiment.

[0055] On the other hand, the present application also provides for using a substance for measuring Zip11 activity and / or expression in the preparation of a reagent, and the reagent is used for identifying the aging state of a subject.

[0056] In some embodiments, the Zip11 activity and / or expression includes Zip11 nucleic acid expression level and / or activity, and / or Zip11 protein expression level and / or activity.

[0057] In some embodiments, the ISVD specific to CTLA4 includes a heavy chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 20, 22, and 24-32.

[0058] In some embodiments, the ISVD specific to CTLA4 includes a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO: 20.

[0059] In some embodiments, the dimer is a homodimer.

[0060] In some aspects, the linker includes the amino acid sequence set forth in any one of SEQ ID NOs: 33-34.

[0061] In some embodiments, one or both of the two polypeptide chains include the amino acid sequence set forth in any one of SEQ ID NOs: 40-43, 46, 48, and 50.

[0062] In some embodiments, one or both of the two polypeptide chains include the amino acid sequence set forth in SEQ ID NO: 40.

[0063] In some embodiments, the dimer can block the binding of PD-L1 to PD-1.

[0064] In some embodiments, the dimer can block the binding of PD-L1 to CD80.

[0065] In some embodiments, the dimer can block the binding of CTLA4 to CD80.

[0066] In some embodiments, the dimer can block the binding of CTLA4 to CD86.

[0067] In some embodiments, the Her2 inhibitor can inhibit human Her2.

[0068] In some embodiments, the Her2 inhibitor is a Her2 antibody or an antigen-binding portion thereof and / or a conjugate thereof.

[0069] In some embodiments, the Her2 antibody is selected from the group consisting of pertuzumab, trastuzumab, and margetuximab.

[0070] In some embodiments, the conjugate is selected from the group consisting of DS8201a and T-DM1.

[0071] In some embodiments, the Her2 inhibitor is a bispecific antibody or an antigen-binding portion thereof and can bind to different epitopes of human Her2.

[0072] In some embodiments, the Her2 inhibitor is a bispecific antibody or an antigen-binding portion thereof, and the bispecific antibody or an antigen-binding portion thereof has a common light chain, and the common light chain refers to two light chains having the same sequence.

[0073] In some embodiments, its heavy chains can correctly associate with the respective light chains under physiological conditions or during in vitro protein expression.

[0074] In some embodiments, the common light chain can associate with the heavy chain of pertuzumab and the heavy chain of trastuzumab, respectively.

[0075] In some embodiments, the common light chain is selected from the light chain of pertuzumab, the light chain of trastuzumab, or a mutant thereof.

[0076] In some embodiments, the sequence of the variable region of the common light chain comprises a sequence selected from the sequences shown at amino acid positions 1 to 107 of SEQ ID NOs: 65 to 70.

[0077] In some embodiments, the heavy chain variable regions are the heavy chain variable regions of pertuzumab and trastuzumab, respectively. For example, the heavy chain variable regions comprise the sequences set forth in SEQ ID NOs: 87 and 88, respectively.

[0078] In some embodiments, the Fc fragment sequences of the heavy chains comprise the sequences set forth in SEQ ID NOs: 89 and 90, respectively.

[0079] In some embodiments, the two heavy chains comprise the sequences set forth in SEQ ID NOs: 83 and 84, respectively.

[0080] In some embodiments, the Her2 inhibitor is administered at a dose of 0.01 mg / kg to 100 mg / kg.

[0081] In some embodiments, the Her2 inhibitor is administered at a dose of 20 mg / kg to 30 mg / kg.

[0082] In some embodiments, the immune checkpoint inhibitor is administered at a dose of 0.01 mg / kg to 100 mg / kg.

[0083] In some embodiments, the immune checkpoint inhibitor is administered at a dose of 3 mg / kg to 5 mg / kg.

[0084] In some embodiments, the Her2 inhibitor is administered at an administration frequency of once every 4 weeks, once every 2 weeks, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, or once every 12 weeks.

[0085] In some embodiments, the Her2 inhibitor is administered once every two weeks or once every three weeks, or as a loading dose.

[0086] In some embodiments, the immune checkpoint inhibitor is administered at a dosing frequency of once every four weeks, once every two weeks, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, or once every twelve weeks.

[0087] In some embodiments, the immune checkpoint inhibitor is administered once every two weeks or once every three weeks.

[0088] In some embodiments, the Her2 inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally.

[0089] In some embodiments, the Her2 inhibitor is administered by intravenous administration.

[0090] In some embodiments, the immune checkpoint inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally.

[0091] In some embodiments, the immune checkpoint inhibitor is administered by intravenous administration.

[0092] In some embodiments, the tumor is selected from the group consisting of solid tumors and hematological tumors.

[0093] In some embodiments, the tumor is selected from the group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, biliary tract cancer, colorectal cancer, and urothelial cancer.

[0094] In some embodiments, the tumor is selected from the group consisting of Her2-abnormal solid tumors and Her2-positive cancers.

[0095] In some embodiments, the subject is administered an anti-Her2 antibody and / or an anti-PD1 agent. In some embodiments, the anti-Her2 antibody includes trastuzumab.

[0096] In some embodiments, the subject is administered chemotherapy.

[0097] In some embodiments, the chemotherapy includes primary chemotherapy and / or secondary chemotherapy. In some embodiments, the secondary chemotherapy includes monotherapy with paclitaxel + ramucirumab, paclitaxel, docetaxel, and / or irinotecan.

[0098] In another form, the present disclosure provides a pharmaceutical composition including an effective amount of the immune checkpoint inhibitor of the present disclosure, an effective amount of the Her2 inhibitor, and optionally a pharmaceutically acceptable excipient.

[0099] In another form, the present disclosure provides the use of a pharmaceutical composition in the preparation of a medicament for treating a tumor of a subject in need thereof.

[0100] In some embodiments, the pharmaceutical composition is administered at an administration frequency of once every four weeks, once every two weeks, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, or once every twelve weeks.

[0101] In some embodiments, the pharmaceutical composition is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, by inhalation, intrathecally, intraventricularly, or intranasally.

[0102] In some embodiments, the pharmaceutical composition is administered at a dose of 0.01 mg / kg to 100 mg / kg.

[0103] In some embodiments, the tumor is selected from the group consisting of solid tumors and hematological tumors.

[0104] In some embodiments, the tumor is selected from the group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, biliary tract cancer, colorectal cancer, and urothelial cancer.

[0105] In some embodiments, the tumor is selected from the group consisting of Her2-abnormal solid tumors and Her2-positive cancers.

[0106] In some embodiments, a subject suffering from the tumor is administered an anti-Her2 antibody and / or an anti-PD1 agent. In some embodiments, the anti-Her2 antibody includes trastuzumab.

[0107] In some embodiments, the subject is administered chemotherapy.

[0108] In some embodiments, the chemotherapy includes primary chemotherapy and / or secondary chemotherapy. In some embodiments, the secondary chemotherapy includes monotherapy with paclitaxel + ramucirumab, paclitaxel, docetaxel, and / or irinotecan.

[0109] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description. Here, only exemplary embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other different embodiments, and some details thereof can be modified in various obvious respects without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

Advantages of the Invention

[0110] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

[0111] The novel features of the present invention are particularly described in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by referring to the following detailed description that describes exemplary embodiments in which the principles of the present invention are employed, and the accompanying drawings (also the "FIG" and "FIG" herein).

[0112]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0113] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, changes, and substitutions can occur to those skilled in the art. It should be understood that various alternatives to the embodiments of the present invention described herein can be used.

[0114] As used herein, the term "antibody" generally refers to an immunoglobulin or a fragment or derivative thereof, and includes any polypeptide containing an antigen-binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and graft antibodies. For the purposes of the present disclosure, unless otherwise modified by the term "intact", such as "intact antibody", the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, etc., and other antibody fragments that retain the antigen-binding function, i.e., the ability to specifically bind to, for example, CTLA-4 or PD-L1. Typically, such fragments contain an antigen-binding domain.

[0115] As used herein, the term "variable region" or "variable domain" of an antibody generally refers to the amino-terminal domain of an antibody heavy or light chain. The variable domains of the heavy and light chains can be designated "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (when compared to other antibodies of the same class) and contain the antigen-binding site.

[0116] As used herein, the term "variable" usually refers to the fact that certain segments of the variable domain vary widely in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for a particular antigen. However, variability is not evenly distributed throughout the span of the variable domain. Instead, it is concentrated in three segments called hypervariable regions (CDRs or HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, mostly adopt a β-sheet structure, are linked by three CDRs, form loops that connect the β-sheet structures and in some cases form part of the β-sheet structures. The CDRs within each chain are held together in close proximity by the FR regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions such as the involvement of the antibody in antibody-dependent cell cytotoxicity.

[0117] As used herein, the terms "CDR", "HVR" or "HV" generally refer to regions of antibody variable domains that are hypervariable and / or form structurally defined loops in the sequence. Generally, an antibody contains six CDRs; three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3). The ISVDs of the present disclosure may contain only three CDRs (e.g., VH, HCDR1, HCDR2, and HCDR3). In natural antibodies, HCDR3 and LCDR3 show the greatest diversity among the six CDRs, and in particular, HCDR3 is thought to play a unique role in conferring fine specificity to the antibody. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, natural camel antibodies consisting of only heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0118] A number of CDR depictions are used and are included herein. Kabat complementarity determining regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM CDRs represent a compromise between Kabat HVRS and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0119] The "contact" CDRs are based on the analysis of available complex crystal structures. The residues of these CDRs are shown in Table 1:

Table 1

[0120] CDRs can include "extended CDRs" as follows. In VL, they are 24-36 or 24-34 (LCDR1), 46-56 or 50-56 (LCDR2), and 89-97 or 89-96 (LCDR3), and in VH, they are 26-35 (HCDR1), 50-65 or 49-65 (HCDR2), and 93-102, 94-102 or 95-102 (HCDR3). The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0121] The expression "Kabat variable domain residue numbering" or "Kabat amino acid position numbering" and variations thereof generally refer to the numbering system used by Kabat et al. for the heavy chain variable domain or light chain variable domain of the dimer / polypeptide chain editing described above. The Kabat numbering of residues can be determined for a given polypeptide by aligning it in regions of homology between the "standard" Kabat-numbered sequence and the polypeptide sequence.

[0122] The "framework" or "FR" residues are variable domain residues other than the CDR residues as defined herein. The "human consensus framework" or "acceptor human framework" is a framework that represents the amino acid residues that are most commonly present in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is the subgroup shown in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). By way of example, for VL, the subgroup may be subgroup κI, κII, κIII or κIV as in Kabat et al., supra. Further, for VH, the subgroup may be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra. Alternatively, the human consensus framework may be derived from the above when human framework residues are selected based on their homology to a donor framework sequence by aligning the donor framework sequence with a collection of various human framework sequences. A receptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may contain the same amino acid sequence or may contain existing amino acid sequence variations. In some embodiments, the number of existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0123] As used herein, the terms "homology", "homologous" or "sequence identity" generally refer to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a program (e.g., Emboss Needle or BestFit) to determine sequence identity, similarity or homology between two different amino acid sequences, default settings may be used, or an appropriate scoring matrix such as blosum45 or blosum80 may be selected to optimize the identity, similarity or homology score. In some embodiments, homologous polynucleotides hybridize under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity with a reference sequence. Homologous polypeptides may have at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity with each other when the sequences of equal length are optimally aligned.

[0124] The term "percent (%) sequence identity", when used in the context of the polypeptide sequences identified herein, generally refers to the percentage of amino acid residues or nucleotides in a query sequence that are identical to the amino acid residues or nucleotides of a second reference polypeptide sequence or a portion thereof, after aligning the sequences, optionally introducing gaps to achieve maximum percent sequence identity, and not considering conservative substitutions as part of sequence identity. Alignments for the purpose of determining percent amino acid / nucleotide sequence identity can be achieved in a variety of ways within the skill of the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithms necessary to achieve the maximum alignment over the full length of the sequences being compared. Percent identity can be measured over the length of the entire defined polypeptide / polynucleotide sequence, or over a shorter length, for example, over the length of a fragment taken from a larger defined polypeptide / polynucleotide sequence. It is understood that any fragment length supported by the sequences shown herein can be used to describe the length over which percent identity can be measured, in a table, figure or sequence listing.

[0125] In the present application, the term "bispecific antibody" refers to an antibody that can bind to two antigens or antigen epitopes, respectively, each containing a light chain and a heavy chain of an antibody that can specifically bind to a first antigen or antigen epitope and a light chain and a heavy chain of an antibody that can specifically bind to a second antigen or antigen epitope. In one embodiment, in the bispecific antibody, the antibody light chain that can specifically bind to the first antigen or antigen epitope and the antibody light chain that can specifically bind to the second antigen or antigen epitope have the same sequence. In one embodiment, in the bispecific antibody, the heavy chain of the antibody that can specifically bind to the first antigen or antigen epitope and the heavy chain of the antibody that can specifically bind to the second antigen or antigen epitope have different sequences.

[0126] As used herein, the term "PD-L1" generally refers to programmed cell death ligand 1 protein, its functional variants and / or its functional fragments. PD-L1, also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein encoded by the CD274 gene (in humans). PD-L1 binds to its receptor, programmed cell death protein 1 (PD-1), and is expressed on activated T cells, B cells, and macrophages (Ishida et al., 1992 EMBO J, 11:3887-3395; Okazaki et al., Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science, 2001; 291: 319-22). The complexation of PD-L1 and PD-1 exerts an immunosuppressive effect by inhibiting T cell proliferation and cytokine production of IL-2 and IFN-γ (Freeman et al., Engagement of PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation, J. Exp. Med. 2000, 192:1027-1034; Carter et al., PD-1:PD-L inhibitory pathway affects both CD4(+) and CD8(+) T cells and is overcome by IL-2. Eur. J. Immunol. 2002, 32:634-643). For example, the term "PD-L1" can include a polypeptide or a fragment thereof that has at least about 85% amino acid sequence identity to NCBI accession number Q9NZQ7 and specifically binds to PD1. The term PD-L1 includes the entire PD-L1 ligand, soluble PD-L1 ligand, and a fusion protein comprising a functionally active portion of the PD-L1 ligand covalently bound to a second moiety, such as a protein domain. Also included in the definition of PD-L1 are variants that have an amino acid sequence different from that of naturally occurring PD-L1 but retain the ability to specifically bind to the receptor PD1.Furthermore, the definition of PD-L1 also includes variants that enhance the biological activity of PD1. The PD-L1 sequence is known in the art and is provided, for example, by GenBank Accession Number 29126. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. For example, the term "PD-L1" also encompasses PD-L1 from other mammals such as rats, mice, rabbits, non-human primates, pigs, or cows. The complete hPD-L1 sequence is described in GenBank Accession No. 29126.

[0127] As used herein, the term "N-terminal IgV domain of human PD-L1" generally refers to the extracellular domain of human PD-L1 located at its N-terminus. Also, the term "N-terminal IgV domain of human PD-L1" may refer to an epitope within said domain. The N-terminal IgV domain of the human PD-L1 protein (including the signal peptide) may contain the amino acid sequence set forth in SEQ ID NO: 64.

[0128] As used herein, the term "CTLA4" generally refers to cytotoxic T lymphocyte-associated protein 4, its functional variants and / or its functional fragments. CTLA4 is an immunosuppressive receptor belonging to the CD28 family. CTLA4 is expressed on T cells (CD4 + cells and CD8 +is expressed only in (cells) and binds to two ligands, CD80 and CD86 (also known as B7-1 and B7-2, respectively). For example, the term "CTLA4" can include a polypeptide or a fragment thereof that has at least about 85% amino acid sequence identity with NCBI accession number AAL 07473.1 and specifically binds to CD80 and / or CD86. The term "CTLA4" includes the entire CTLA4 receptor, its extracellular domain, and a fusion protein consisting of a second moiety, such as a functionally active portion of CTLA4 covalently attached to a protein domain. Also included in the definition of CTLA4 are variants that have an amino acid sequence different from that of naturally occurring CTLA4 but retain the ability to specifically bind to the ligands CD80 and / or CD86. CTLA4 sequences are known in the art and are provided, for example, by GenBank Accession No. 1493. As used herein, the term "CTLA4" includes human CTLA4 (hCTLA4), variants, isoforms, and species homologs of hCTLA4, as well as analogs having at least one common epitope with hCTLA4. For example, the term "CTLA4" also encompasses CTLA4 from other mammals, such as rats, mice, rabbits, non-human primates, pigs, or cows. The complete hCTLA4 sequence is described in GenBank Accession No. 1493.

[0129] As used herein, the term "antibody Fc subunit" generally refers to a component of an antibody Fc domain. For example, an antibody Fc domain can be formed by two or more members, and each member can be considered an Fc subunit. The term "Fc domain" as used herein generally refers to the Fc portion or Fc fragment of an antibody heavy chain. For example, it may refer to the carboxyl-terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof that can bind to an Fc receptor. As is known, each immunoglobulin heavy chain constant region contains four or five domains. The domains are sequentially named as follows: CH1-hinge-CH2-CH3(-CH4). CH4 is present in IgM that does not have a hinge region. Fc domains or Fc subunits useful in the present disclosure may include the CH3 domain. For example, an Fc domain or Fc subunit may include the CH2 domain and the CH3 domain. In some embodiments, the Fc domain or Fc subunit may also include an immunoglobulin hinge region. For example, an Fc domain or Fc subunit may include, or may include from N-terminus to C-terminus, the CH2 domain and the CH3 domain. In another example, an Fc domain or Fc subunit may include, or may be composed of, from N-terminus to C-terminus, an immunoglobulin hinge region, the CH2 domain, and the CH3 domain. The amino acid residue positions within the Fc domain or Fc subunit can be determined according to Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest 5th ed. NIH Publication No. 91-3242.

[0130] As used herein, the term "Fc domain" generally refers to the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The boundaries of the Fc region of an immunoglobulin heavy chain can vary, but the human IgG heavy chain Fc region is typically defined as extending from the amino acid residue at position Cys226, or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody. Thus, a composition of full-length antibodies can include a population of antibodies in which all K447 residues have been removed, a population of antibodies in which the K447 residues have not been removed, and a population of antibodies having a mixture of antibodies with and without the K447 residue. Native sequence Fc regions suitable for use in the antibodies of the present invention include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0131] Unless otherwise indicated herein, the numbering of residues in immunoglobulin chains is as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., the EU Index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). The "EU index" in Kabat refers to the residue numbers of the human IgG1 EU antibody.

[0132] As used herein, the term "dimer" generally refers to a macromolecular complex formed by two, usually non-covalently, monomer units. Each monomer unit can be a macromolecule such as a polypeptide chain or a polynucleotide. As used herein, the term "homodimer" generally refers to a dimer composed of or formed by two substantially identical monomers, such as two substantially identical polypeptide chains. In some cases, the two monomers of a homodimer may differ at one or more regions or positions, but such differences do not cause a significant change in the function or structure of the monomer. For example, one of ordinary skill in the art would consider the differences between the two monomers to have little or no biological and / or statistical significance in the context of the biological properties considered in the present disclosure. The structural / compositional differences between the two monomers may be, for example, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, or less.

[0133] As used herein, the term "fused" or "fusion" generally refers to a covalent bond between two polypeptides. The polypeptides are typically joined directly to each other or via an amino acid linker through a peptide bond. Optionally, the peptides can be joined via non-peptide covalent bonds known to those of ordinary skill in the art.

[0134] As used herein, the term "fusion protein" generally refers to a polypeptide consisting of or alternatively comprising the amino acid sequence of a polypeptide that is directly or indirectly (e.g., via a linker) fused to the amino acid sequence of a heterologous polypeptide (i.e., a polypeptide unrelated to the former polypeptide or its domain).

[0135] As used herein, the term "immunoglobulin single variable domain (ISVD)" generally refers to fragments such as antigen-binding domains or VHH domains or VH or VL domains, respectively. The terms antigen-binding molecule or antigen-binding protein are used interchangeably and also include the term nanobody. The immunoglobulin single variable domain is further a light chain variable domain sequence (e.g., VL-sequence), or a heavy chain variable domain sequence (e.g., VH-sequence), and more specifically can be a heavy chain variable domain sequence derived from a conventional 4-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody. Thus, the immunoglobulin single variable domain can be a domain antibody, or an immunoglobulin sequence suitable for use as a domain antibody, a single domain antibody, a single domain antibody, or an immunoglobulin sequence suitable for use as a single domain antibody, a "dAb", or an immunoglobulin sequence suitable for use as a dAb, or a nanobody including but not limited to a VHH sequence. The immunoglobulin single variable domain includes fully human, humanized, otherwise sequence-optimized, or chimeric immunoglobulin sequences. The immunoglobulin single variable domain and the structure of the immunoglobulin single variable domain can, however, without being limited thereto, be considered to be composed of four framework regions or "FRs", which are referred to in the art and herein as "framework region 1" or "FR1"; "framework region 2" or "FR2"; "framework region 3" or "FR3"; and "framework region 4" or "FR4", respectively, and these framework regions are interrupted by three complementarity-determining regions or "CDRs", which are referred to in the art as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3", respectively.

[0136] As used herein, the term "humanized" generally refers to an antibody or fragment thereof in which some, most, or all of the amino acids outside the CDR domains of a non-human antibody have been replaced with the corresponding amino acids derived from a human immunoglobulin. For example, in a humanized form of an antibody, some, most, or all of the amino acids outside the CDR domains are replaced with amino acids derived from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDR regions are not altered. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are tolerated as long as the antibody's ability to bind its particular antigen / epitope is not impaired. A humanized antibody can retain the same antigen specificity as the original antibody.

[0137] As used herein, the term "epitope" or "antigenic determinant" generally refers to the site on an antigen to which an antibody binds. An epitope can be formed from adjacent amino acids (linear epitope) or from non-adjacent amino acids juxtaposed by the three-dimensional folding of a protein (conformational epitope). An epitope formed from adjacent amino acids is typically retained upon exposure to a denaturing solvent, while an epitope formed by three-dimensional folding is typically lost upon treatment with a denaturing solvent. An epitope typically contains at least 3, more commonly at least 5 or 8-10, amino acids in a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed (1996).

[0138] As used herein, the term "conformational epitope" generally refers to non - adjacent amino acid residues of an antigen (such as the PD - L1 antigen) juxtaposed by the tertiary folding of a protein. These discontinuous amino acid residues may come together on the surface when the polypeptide chain folds to form the native protein. Conformational epitopes include, but are not limited to, functional epitopes.

[0139] In this application, the 20 conventional amino acids and their abbreviations follow conventional rules. As a reference, it is described in Immunology - A Synthesis (Edition II, E.S. Golub and D.R. Gren, Eds. Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0140] As used herein, the term "functional epitope" generally refers to the amino acid residues of an antigen that contribute energetically to antibody binding, i.e., form an "energy epitope". Any single mutation of an energetically contributing residue of the antigen to alanine disrupts antibody binding, and as a result, the relative K D ratio (K D mutant / K D wild - type) can be, for example, more than 3 - fold, more than 4 - fold, more than 6 - fold, more than 10 - fold, more than 20 - fold, more than 30 - fold, more than 40 - fold, more than 50 - fold, more than 60 - fold, more than 70 - fold, more than 80 - fold, more than 90 - fold, more than 100 - fold, more than 150 - fold, more than 200 - fold, or more.

[0141] As used herein, the term "extracellular domain" generally refers to a portion of a protein (such as a membrane protein like a receptor) that protrudes from a cell organelle and / or the outer membrane of a cell. When the polypeptide chain passes through the bilayer several times, the extracellular domain consists of loops intertwined with the membrane. The extracellular domain can recognize and respond to specific ligands.

[0142] As used herein, the term "linker" generally refers to a synthetic amino acid sequence that connects or joins two polypeptide sequences, for example, that joins two polypeptide domains. A linker may connect two amino acid sequences via a peptide bond. In some embodiments, the linkers of the present disclosure linearly link a biologically active site to a second site. For example, a peptide linker may be non-immunogenic and flexible, such as one consisting of serine and glycine sequences or a repeat of Ala-Ala-Ala. Depending on the particular configuration of the dimer, the peptide linker may, for example, be composed of the following. It may contain 3 to 30 (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30) amino acid residues.

[0143] The term "N-terminus" may be used interchangeably with "N-terminal" and, as used herein, these generally refer to the amino terminus / end of a polypeptide chain.

[0144] The term "C-terminus" may be used interchangeably with "C-terminal" and, as used herein, these generally refer to the carboxyl terminus / end of a polypeptide chain.

[0145] As used herein, the term "about" generally refers to variations within the normal tolerances in the art and generally within ±10%, for example, within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless otherwise apparent from the context, all numerical values provided herein are modified by the term about.

[0146] As used herein, the terms "combination," "simultaneous administration," or "co-administration" generally refer to the administration of one active ingredient (e.g., a dimer) together with another active ingredient (e.g., an immune checkpoint inhibitor). The administration of one active ingredient can be performed as one formulation or as two separate formulations (e.g., the dimer and one of the immune checkpoint inhibitors). Co-administration can be simultaneous or sequential in any order.

[0147] The term "specifically binds" or "specific" as used herein generally refers to a measurable and reproducible interaction, such as binding between a target and an antibody, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. In one embodiment, the extent of binding of the antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a binding affinity of <1x10 -6 M, <1x10 -7 M, <1x10 -8 M, <1x10 -9 M, or <1x10 -10 Dissociation constant of M (K D ). In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding can include, but does not require, exclusive binding.

[0148] The basic four-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light chains (L chains) and two identical heavy chains (H chains). IgM antibodies consist of five basic four-chain units and an additional polypeptide called the J chain, and have ten antigen-binding sites. IgA antibodies consist of two to five basic four-chain units and can polymerize by binding to the J chain to form a multivalent aggregate. In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is bound to the H chain by one covalent disulfide bond, while the two H chains are bound to each other by one or more disulfide bonds depending on the heavy chain isotype. Also, the H chain and L chain each have intra-chain disulfide bonds (SS) at regular intervals. The H chain has a variable domain (VH) at the N-terminus, the α chain and γ chain each have three constant domains (CH), and μ and ε have four CH domains. The L chain has a variable domain (VL) at the N-terminus and a constant domain (CH) at the other end. VL aligns with VH, and CL aligns with the first constant domain (CHI) of the heavy chain. Certain amino acid residues are thought to form the interface between the light chain variable domain and the heavy chain variable domain. One antigen-binding site is formed by the pairing of VH and VL. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6. The L chains of vertebrate species are classified into one of two clearly distinct types called kappa and lambda based on the amino acid sequence of the constant domain. Immunoglobulins are assigned to different classes or isotypes depending on the amino acid sequence of the constant domain of the heavy chain (CH). Immunoglobulins are classified into five classes. IgA, IgD, IgE, IgG, and IgM have heavy chains called α, δ, ε, γ, and μ, respectively.The y and a classes are further classified into subclasses based on relatively small differences in the CH sequences and functions. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.

[0149] In this application, the term "antigen-binding portion" of an antibody refers to one or more portions of a full-length antibody, and the antigen-binding portion maintains the ability to bind to the same antigen (such as Her2) as that bound by the antibody and competes with the full-length antibody for specific binding to the antigen. Generally, see Fundamental Immunology, Ch.7 (Paul W. ed. edition II Raven Press, N.Y. 1989), which is hereby incorporated by reference in its entirety and for all purposes. The antigen-binding portion can be produced using recombinant DNA technology or by enzymatic or chemical cleavage of a full-length antibody. In some cases, the antigen-binding portion includes polypeptides such as Fab, Fab’, F(ab’)2, Fd, Fv, dAb, complementarity-determining region (CDR) fragments, single-chain antibodies (such as scFv), chimeric antibodies, and diabodies, and includes at least a portion of an antibody that sufficiently confers specific antigen-binding ability to the polypeptide. The antigen-binding portion of an antibody (such as the above antibody fragments) can be obtained from a given antibody (such as monoclonal antibody 2E12) using conventional techniques known to those skilled in the art (such as recombinant DNA technology or enzymatic or chemical cleavage processes) and is screened for its specificity in the same process as screening for a full-length antibody.

[0150] As used herein, the term "polypeptide chain" generally refers to a macromolecule containing two or more covalently bonded peptides. The peptides within a polypeptide chain may be linked to each other via peptide bonds. Each polypeptide chain may include one N-terminus or amino terminus and one C-terminus or carboxy terminus.

[0151] As used herein, the term "CD80" generally refers to the ligand for CD28 / CTLA4, also known as B7.1, its functional variants and / or its functional fragments. CD80 is generally expressed on the surface of specialized antigen-presenting cells (APCs). For example, the term "CD80" can include a polypeptide or a fragment thereof that has at least about 85% amino acid sequence identity to NCBI Accession No. P33681 and specifically binds to CTLA4. The definition of CD80 includes variants that have an amino acid sequence different from that of naturally occurring CD80 but retain the ability to specifically bind to CTLA4. Further included in the definition of CD80 are variants that enhance the biological activity of CTLA4. The sequence of CD80 is known in the art and is provided, for example, by GenBank Accession Numbers P33681. As used herein, the term "CD80" includes human CD80 (hCD80), variants, isoforms, and species homologs of hCD80, and analogs having at least one common epitope with hCD80. For example, the term "CD80" also encompasses CD80 from other mammals such as rats, mice, rabbits, non-human primates, pigs, or cows and other species. The complete hCD80 sequence is described in GenBank Accession No. P33681.

[0152] As used herein, the term "CD86" generally refers to the ligand for CD28 / CTLA4, also known as B7.2, its functional variants and / or its functional fragments. CD86 is generally expressed on the surface of specialized antigen-presenting cells (APCs). For example, the term "CD86" can include a polypeptide or a fragment thereof that has at least about 85% amino acid sequence identity to NCBI Accession No. P42081 and specifically binds to CTLA4. Also included in the definition of CD86 are variants that have an amino acid sequence different from that of naturally occurring CD86 but retain the ability to specifically bind to CTLA4. Further included in the definition of CD86 are variants that enhance the biological activity of CTLA4. CD86 sequences are known in the art and are provided, for example, by GenBank Accession Numbers U04343. As used herein, the term "CD86" includes human CD86 (hCD86), variants, isoforms, and species homologs of hCD86, as well as analogs having at least one common epitope with hCD86. For example, the term "CD86" also encompasses CD86 from other mammals, such as rats, mice, rabbits, non-human primates, pigs, or cows, and other species. The complete hCD86 sequence is described in GenBank Accession No. U04343.

[0153] As used herein, the term "PD1" generally refers to the programmed cell death-1 receptor, also known as CD279, its functional variants and / or its functional fragments. PD1 is generally expressed on T cells, B cells, natural killer T cells, activated monocytes and dendritic cells (DCs), and PD1 can bind to its ligands PD-L1 and PD-L2. For example, the term "PD1" can include a polypeptide or a fragment thereof that has at least about 85% amino acid sequence identity to NCBI Accession No P42081 and specifically binds to PD-L1. Also included in the definition of PD1 are variants that have an amino acid sequence different from that of naturally occurring PD1 but retain the ability to specifically bind to PD-L1. Further included in the definition of PD1 are variants that enhance the biological activity of PD-L1. PD1 sequences are known in the art and are provided, for example, by GenBank Accession Number Q15116.3. The term "PD1" as used herein includes human PD1 (hPD1), variants, isoforms, and species homologs of hPD1, as well as analogs having at least one common epitope with hPD1. For example, the term "PD1" also encompasses PD1 from other mammals such as rats, mice, rabbits, non-human primates, pigs, or cows, etc. The complete hPD1 sequence is described in GenBank Accession No. Q15116.3.

[0154] As used herein, the term "blocking" generally refers to the inhibition or reduction of binding activity between a molecule and its specific binding partner, such as between a ligand and its specific receptor.

[0155] As used herein, the terms "blocking antibody" and "antagonist antibody" generally refer to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. In some embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. The PD-L1-specific ISVDs or CTLA4-specific ISVDs of the present disclosure can be blocking or antagonistic ISVDs. For example, the PD-L1-specific ISVD of the present disclosure can block the interaction between PD-L1 and its receptor PD-1 so as to restore a functional response from a dysfunctional state of T cells to antigen stimulation, and thus can block signal transduction via PD-1. The CTLA4-specific ISVD of the present disclosure can block the interaction between CTLA4 and CD80 / CD86 so as to restore a functional response from a dysfunctional state of T cells to antigen stimulation, and thus can block signal transduction via CTLA4.

[0156] As used herein, the terms "cross-competing entity for binding", "cross-competition", "cross-inhibition", "cross-blocking", and "cross-block" are used interchangeably and generally refer to the ability of an antibody or fragment thereof to directly or indirectly interfere with binding through allosteric modulation to the target / antigen (e.g., PD-L1 or CTLA4, respectively) of another antibody of the invention (e.g., a PD-L1-specific ISVD or a CTLA4-specific ISVD of the present disclosure). Whether an antibody or fragment thereof can interfere with the binding of another antibody to a target, and thus can be said to cross-block or cross-compete according to the present invention, can be determined using a competitive binding assay. One particularly preferred quantitative cross-competition assay measures competition in terms of binding to a target between a labeled (e.g., His-tagged, biotinylated, or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof using a FACS-based or AlphaScreen-based approach. Generally, a cross-competing antibody or fragment thereof binds to the target in a cross-competition assay such that, for example, during the assay and in the presence of a second antibody or fragment thereof, the recorded substitutions of the immunoglobulin single variable domain or polypeptide according to the present invention are up to 100% of the maximum theoretical substitution (e.g., substitution by a cold (e.g., unlabeled) antibody or fragment thereof that needs to be cross-blocked) by the potential cross-blocking antibody or fragment thereof to be tested, present in a given amount. Preferably, the cross-competing antibody or fragment thereof has recorded substitutions between 10% and 100%, such as between 50% and 100%.

[0157] As used herein, the term "substantially reduced" or "substantially different" generally refers to a sufficiently high degree of difference between two numerical values (generally, one related to a molecule and the other related to a reference / comparator molecule) such that one of ordinary skill in the art would conclude that the difference between the two values is significant relative to the values (e.g., K Dwould be considered a statistically significant difference within the context of the biological property measured by the value). The difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and / or greater than about 50% as a function of the value of the reference / comparison molecule.

[0158] As used herein, the terms “substantially similar” or “substantially the same” generally refer to a sufficiently high degree of similarity between two numerical values (e.g., those associated with a molecule of the present disclosure and those associated with a reference / comparison molecule), such that one of ordinary skill in the art would consider the difference between the values to have little or no biological and / or statistical significance within the context of the biological property (e.g., K D value) measured by the values. The difference between the two values is, for example, about 50% or less, about 40% or less, about 30% or less, about 20% or less, and / or about 10% or less as a function of the reference value / comparison value.

[0159] In the present disclosure, the amino acid sequences or nucleotide sequences described by a specific SEQ ID NO. also include homologs or variants thereof having substantially the same function / characteristics. For example, sequences having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity; and / or variants having an addition, deletion, or substitution of one or more (e.g., a small number such as 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2) amino acids or nucleotides.

[0160] As used herein, the term “tumor” generally refers to a clinically measurable degree of tumor growth or metastasis. The tumor can be a solid tumor, a hematological tumor, or a lymphoma. For example, the tumor can be selected from lung cancer (such as non-small cell lung cancer), breast cancer (such as triple negative breast cancer), kidney cancer (such as renal cell carcinoma), melanoma, cervical cancer, uterine cancer, pancreatic cancer, peritoneal cancer, ovarian cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, biliary tract cancer, urothelial cancer, and colorectal cancer. The tumor can be a progressive tumor or a metastatic tumor.

[0161] As used herein, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, goats, rabbits, mice, rats, or monkeys.

[0162] As used herein, the term "treat" generally means having a therapeutic effect and at least partially alleviating or eliminating an abnormal condition in a living body. The term "treat" as used herein refers to improving the symptoms of a drug condition as compared to a control group not administered the drug in a group of patients administered the drug. The effect of treatment can be monitored by measuring changes or non-changes in cell phenotype changes, cell proliferation changes, tumor size changes, tumor size changes, progressive disease changes, stable disease changes, disease control rate changes, partial response changes, or non-changes. The terms "treat" or "treatment" do not necessarily mean complete cure. Alleviation of undesirable symptoms of a disease to some extent, or slowing of the progression of a disease, can be considered treatment. Furthermore, treatment may in some cases include actions that worsen the overall sense of health or appearance of a patient.

[0163] As used herein, the term "pharmaceutically acceptable excipient" includes, as known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pages 1289 - 1329), any solvent, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antibacterial, antifungal agent), isotonic agent, absorption delaying agent, salt, preservative, drug stabilizer, binder, excipient, disintegrant, lubricant, sweetening agent, flavoring agent, dye, and their analogs and combinations thereof. Its use in therapeutic or pharmaceutical compositions is contemplated, except when any conventional carrier is incompatible with the active ingredient.

[0164] As used herein, the term "effective amount" of a compound generally refers to the amount of a compound of the present invention that induces a biological or medical response in a subject, such as a decrease or inhibition of enzyme or protein activity, improves symptoms, alleviates conditions, delays or retards the progression of a disease, or prevents a disease or the like.

[0165] As used herein, the term "antigen-binding fragment" generally refers to a part of an antibody molecule that contains the amino acids involved in the specific binding between an antibody and an antigen. The part of the antigen that is specifically recognized and bound by an antibody is called an "epitope" as described above herein. As described above, an antigen-binding domain typically can be composed of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but need not be composed of both. For example, an Fd fragment has two VH regions and often retains some of the antigen-binding functions of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include: (1) a Fab fragment, which is a monovalent fragment having VL, VH, CL, and CH1 domains; (2) an F(ab′)2 fragment, which is a divalent fragment in which two Fab fragments are disulfide-bonded at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having VL and VH domains of a single arm of an antibody; (5) a dAb fragment (Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli," Nature 341:544-546 (1989), which is incorporated herein by reference) VH domain; (6) an isolated complementarity-determining region (CDR); and (7) a single-chain Fv (scFv), for example, obtained from an scFv library. The two domains of an Fv fragment, VL and VH, are encoded by separate genes, but these can be joined by a synthetic linker that can be made using recombinant methods such that the VL and VH regions pair to form a single protein chain that forms a monovalent molecule (known as a single-chain Fv (scFv)) (see, for example, Huston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988), which is incorporated herein by reference in its entirety).These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are evaluated for function in the same manner as intact antibodies.

[0166] As used herein, the term "combination" of a first agent and a second agent generally means co - administration of the first agent and the second agent. For example, they may be dissolved or intermixed in the same pharmaceutically acceptable carrier, the second agent may be administered after administration of the first agent, or the first agent may be administered after administration of the second agent. Accordingly, the present disclosure includes methods of combination therapeutic treatment and combination pharmaceutical compositions.

[0167] As used herein, the term "Her2" generally refers to a type I transmembrane protein also known as c - erbB2, ErbB2 or Neu, which belongs to the epidermal growth factor receptor family. In the context of the present disclosure, the term "Her2" also encompasses isoforms including homologs, variants, and splice isoforms of Her2. The term "Her2" further encompasses proteins having sequences of one or more Her2 homologs, variants, and isoforms, and sequences of fragments of the sequences, when the variant protein (including isoforms), homologous protein and / or fragment is recognized by one or more Her2 - specific antibodies provided as, for example, pertuzumab, trastuzumab, and margetuximab. Her2 may be human Her2. The human Her2 gene maps to chromosome position 17q12, and the genomic sequence of the Her2 gene is described in GenBank accession number NG_007503.1. In humans, five Her2 isoforms exist. As used herein, the term "Her2" is used to collectively refer to all Her2 isoforms.

[0168] Dimer

[0169] In one aspect, the present disclosure provides the use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating a tumor in a subject in need of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is capable of specifically binding to PD-L1 and CTLA-4

[0170] In some embodiments, the immune checkpoint inhibitor can be a dimer. The dimer can be formed by two polypeptide chains, each of the two polypeptide chains containing an antibody Fc subunit. For example, the dimer may be composed of two polypeptide chains each containing an antibody Fc subunit, and the antibody Fc subunit of one polypeptide chain may associate with the antibody Fc subunit of the other polypeptide chain to form a dimer. In one example, the two polypeptide chains of the dimer do not fuse with each other (e.g., via a peptide linker or by a peptide bond) to form a single polypeptide chain.

[0171] The dimer can contain two or more immunoglobulin single variable domains (ISVDs). For example, one polypeptide chain of the dimer can contain two or more ISVDs, and the other polypeptide chain of the dimer can contain no ISVDs. In another example, each of the two polypeptide chains can contain one or more ISVDs. In yet another example, each of the two polypeptide chains can be composed of two or more ISVDs.

[0172] At least one of the ISVDs may be specific to PD-L1, and at least one of the ISVDs may be specific to CTLA4. For example, one polypeptide chain of the dimer can include one or more ISVDs specific to PD-L1 and one or more ISVDs specific to CTLA4, and the other polypeptide chain of the dimer can include no ISVDs. In another example, one polypeptide chain of the dimer can include one or more ISVDs specific to PD-L1, and the other polypeptide chain of the dimer can include one or more ISVDs specific to CTLA4. In another example, one polypeptide chain of the dimer can include one or more ISVDs specific to PD-L1 and one or more ISVDs specific to CTLA4, and the other polypeptide chain of the dimer can include one or more ISVDs specific to PD-L1 and / or one or more ISVDs specific to CTLA4.

[0173] One or more ISVDs specific to PD-L1 may be the same or different. One or more ISVDs specific to CTLA4 may be the same or different.

[0174] In some cases, the ISVD specific to PD-L1 does not include any antibody light chain CDRs. In some cases, the ISVD specific to PD-L1 does not include any antibody light chain variable regions. In some cases, the ISVD specific to PD-L1 does not include any antibody light chains or fragments thereof. In some cases, the ISVD specific to PD-L1 includes at least the heavy chain CDR3. In some embodiments, the ISVD specific to PD-L1 includes the heavy chain CDR1. In some cases, the ISVD specific to PD-L1 includes the heavy chain CDR2. In some cases, the ISVD specific to PD-L1 includes the heavy chain variable region. In some cases, the ISVD specific to PD-L1 is an anti-PD-L1 VHH. The ISVD specific to PD-L1 may be humanized.

[0175] In some cases, the ISVD specific for CTLA4 does not contain any antibody light chain CDRs. In some cases, the ISVD specific for CTLA4 does not contain any antibody light chain variable regions. In some cases, the ISVD specific for CTLA4 does not contain any antibody light chains or fragments thereof. In some cases, the ISVD specific for CTLA4 contains at least the heavy chain CDR3. In some cases, the ISVD specific for CTLA4 contains the heavy chain CDR1. In some cases, the ISVD specific for CTLA4 contains the heavy chain CDR2. In some cases, the ISVD specific for CTLA4 contains the heavy chain variable region. In some cases, the ISVD specific for CTLA4 is an anti-CTLA4 VHH. The ISVD specific for CTLA4 may be humanized.

[0176] In some cases, at least one of the two polypeptide chains may contain both an ISVD specific for PD-L1 and an ISVD specific for CTLA4. For example, one of the two polypeptide chains may contain one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4. In another example, each of the two polypeptide chains may contain one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4.

[0177] For one or both of the two polypeptide chains, the ISVD specific for PD-L1 may be fused to the ISVD specific for CTLA4, optionally via a linker. For example, in one or both of the two polypeptide chains, there may be one or more ISVDs specific for PD-L1 and one or more ISVDs specific for CTLA4. If there are two or more ISVDs specific for PD-L1 in a single polypeptide chain, they may be fused to each other (e.g., directly or via a peptide linker), and one or more of them may further be fused to one or more ISVDs specific for CTLA4. If there are two or more ISVDs specific for CTLA4 in a single polypeptide chain, they may be fused to each other (e.g., directly or via a peptide linker), and one or more of them may further be fused to one or more ISVDs specific for PD-L1. One or more linkers (e.g., peptide linkers) may be present between any two ISVDs, e.g., between two ISVDs specific for PD-L1, between two ISVDs specific for CTLA4, or between one ISVD specific for PD-L1 and one ISVD specific for CTLA4.

[0178] For one or both of the two polypeptide chains, the ISVD specific for PD-L1 may be fused, optionally via a linker, to the ISVD specific for CTLA4; and the ISVD specific for CTLA4 may in turn be optionally fused, via a linker, to an antibody Fc subunit. For example, in a single polypeptide chain, the ISVD specific for PD-L1 may be fused directly (e.g., in-frame) or via a linker to the ISVD specific for CTLA4, and the ISVD specific for CTLA4 may be fused directly (e.g., in-frame) or via a linker to an antibody Fc subunit. If there are two or more ISVDs specific for PD-L1 and / or one or more ISVDs specific for CTLA4 present in one polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused to each other directly or via a linker in any order, and at least one ISVD specific for CTLA4 may be fused directly (e.g., in-frame) or via a linker to an antibody Fc subunit. For example, for one or both of the two polypeptide chains, the C-terminus of the ISVD specific for PD-L1 may be fused, optionally via a linker, to the N-terminus of the ISVD specific for CTLA4; and the C-terminus of the ISVD specific for CTLA4 may be fused, optionally via a linker, to the N-terminus of an antibody Fc subunit. For example, in a single polypeptide chain, the C-terminus of one of the ISVDs specific for PD-L1 may be fused directly (e.g., in-frame) or via a linker to the N-terminus of one of the ISVDs specific for CTLA4, and the C-terminus of one of the ISVDs specific for CTLA4 may be fused directly (e.g., in-frame) or via a linker to the N-terminus of an antibody Fc subunit.In one example, when one or more ISVDs specific for PD-L1 and / or one or more ISVDs specific for CTLA4 are present in a single polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused to each other in any order, either directly or via a linker, but the C-terminus of at least one ISVD specific for PD-L1 may be fused to the N-terminus of at least one ISVD specific for CTLA4, either directly (e.g., in-frame) or via a linker, and the C-terminus of at least one ISVD specific for CTLA4 may be fused to the N-terminus of an antibody Fc subunit, either directly (e.g., in-frame) or via a linker.

[0179] For one or both of the two polypeptide chains, the ISVD specific for CTLA4 may be fused, optionally via a linker, to the ISVD specific for PD-L1; and the ISVD specific for PD-L1 may then be fused, optionally via a linker, to the antibody Fc subunit. For example, in a single polypeptide chain, the ISVD specific for CTLA4 may be fused directly (e.g., in-frame) or via a linker to the ISVD specific for PD-L1, and the ISVD specific for PD-L1 may be fused directly (e.g., in-frame) or via a linker to the antibody Fc subunit. If there are two or more ISVDs specific for PD-L1 and / or two or more ISVDs specific for CTLA4 in one polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused to each other directly or via a linker in any order, and at least one ISVD specific for PD-L1 may be fused directly (e.g., in-frame) or via a linker to the antibody Fc subunit. For example, for one or both of the two polypeptide chains, the C-terminus of the ISVD specific for CTLA4 may be fused, optionally via a linker, to the N-terminus of the ISVD specific for PD-L1; and the C-terminus of the ISVD specific for PD-L1 may be fused, optionally via a linker, to the N-terminus of the antibody Fc subunit. For example, in a single polypeptide chain, the C-terminus of one of the ISVDs specific for CTLA4 may be fused directly (e.g., in-frame) or via a linker to the N-terminus of one of the ISVDs specific for PD-L1, and the C-terminus of one of the ISVDs specific for PD-L1 may be fused directly (e.g., in-frame) or via a linker to the N-terminus of the antibody Fc subunit.In one example, when one or more ISVDs specific for PD-L1 and / or one or more ISVDs specific for CTLA4 are present in one polypeptide chain, the ISVDs specific for PD-L1 and the ISVDs specific for CTLA4 may be fused to each other in any order, either directly or via a linker, provided that the C-terminus of at least one ISVD specific for CTLA4 is fused to the N-terminus of at least one ISVD specific for PD-L1, either directly (e.g., in-frame) or via a linker, and the C-terminus of at least one ISVD specific for PD-L1 is fused to the N-terminus of an antibody Fc subunit, either directly (e.g., in-frame) or via a linker.

[0180] The linker (e.g., peptide linker) used in the present application (e.g., as constituted by the dimer of the present application) may be a synthetic amino acid sequence that links or concatenates two polypeptide sequences, for example, via a peptide bond. For example, the peptide linker may contain 1 to 10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids), 1 to 15 amino acids (e.g., 1 to 10, 11, 12, 13, 14 or 15 amino acids), 1 to 20 amino acids (e.g., 1 to 15, 16, 17, 18, 19 or 20 amino acids), 1 to 30 or more amino acids (e.g., 1 - 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids). For example, the peptide linker may contain the amino acid sequence set forth in any one of SEQ ID NOs: 33 - 34.

[0181] The antibody Fc subunit may be derived from an IgG Fc subunit. For example, IgG can be selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In some embodiments, IgG is human IgG1 and the IgG Fc subunit is a human IgG1 Fc subunit. In some embodiments, the Fc subunit comprises an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 35, 38, and 39. For example, the Fc subunit may comprise an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid deletions, insertions, and / or substitutions in the amino acid sequence set forth in any of SEQ ID NOs: 35, 38, and 39.

[0182] In some embodiments, the Fc subunit may be a variant of an IgG Fc subunit (e.g., a variant of a human IgG1 Fc subunit). For example, the variant may comprise one or more amino acid mutations that enhance or decrease ADCC or CDC activity. As another example, the variant may comprise one or more amino acid mutations that affect FcRn binding activity and / or the half-life of a molecule comprising the variant. As yet another example, the variant may comprise one or more amino acid mutations that affect the interaction (e.g., association) between two or more Fc subunits (or Fc monomers) and / or increase or decrease the efficiency of Fc heterodimer formation. For example, the variant may comprise one or more amino acid substitutions as described in CN 102558355 A, CN 103388013 A, CN 105820251 A, or CN 106883297 A, each of which is incorporated herein by reference.

[0183] The ISVD specific for PD-L1 may specifically bind to human PD-L1. For example, the ISVD specific for PD-L1 may specifically bind to an epitope within the extracellular domain of human PD-L1. Such epitopes are known in the art, as shown, for example, by Gang Hao et al., J. Mol. Recognit.. 2015; 28:269-276, Zhang et al., Oncotarget. 2017 Oct; 08 (52):90215-90224, and Zhang et al., Cell Discov. 2017 Mar 7; 3:17004.

[0184] For example, an ISVD specific to PD-L1 can bind to the N-terminal IgV domain of human PD-L1. The N-terminal IgV domain of human PD-L1 (including the signal peptide) can contain the amino acid sequence set forth in SEQ ID NO: 64. In the present disclosure, an ISVD specific to PD-L1 can bind to residues 154, Y56, E58, Q66, and / or R113 of the human PD-L1 N-terminal IgV domain. In certain embodiments, an ISVD specific to PD-L1 can bind to residues 154, Y56, E58, Q66, and R113 of the human PD-L1 N-terminal IgV domain (e.g., amino acid residues 154, Y56, E58, Q66, and / or R113 of SEQ ID NO: 64). An ISVD specific to PD-L1 can further bind to residues D61, N63, V68, M115, S117, Y123, and / or R125 of the human PD-L1 N-terminal IgV domain (e.g., amino acid residues D61, N63, V68, M115, S117, Y123, and / or R125 of SEQ ID NO: 64). In some cases, an ISVD specific to PD-L1 can bind to the conformational epitope of the human PD-L1 N-terminal IgV domain, and the conformational epitope can include residues 154, Y56, E58, Q66, and / or R113 of the human PD-L1 N-terminal IgV domain (e.g., amino acid residues 154, Y56, E58, Q66, and / or R113 of SEQ ID NO: 64).In some cases, an ISVD specific for PD-L1 can bind to a conformational epitope of the human PD-L1 N-terminal IgV domain, which may include amino acid residues 154, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123, and / or R125 of the human PD-L1 N-terminal IgV domain (e.g., amino acid residues 154, Y56, E58, Q66, R113, D61, N63, V68, M115, S117, Y123, and / or R125 of SEQ ID NO: 64).

[0185] The ISVD specific to PD-L1 of the present disclosure (e.g., PD-L1 ISVD-9 and its humanized variants) binds to the N-terminal IgV domain of human PD-L1. Taking PD-L1 ISVD-9 as an example, the residue Phe101 (SEQ ID NO: 6) of PD-L1 ISVD-9 interacts with Tyr 56 of the human PD-L1 N-terminal IgV domain. When Tyr 56 of the human PD-L1 N-terminal IgV domain was substituted with Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 decreased by more than 200-fold. When Ile 54 of the human PD-L1 N-terminal IgV domain was substituted with Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 decreased by approximately 40-fold. The residue Asp 99 (SEQ ID NO: 6) of PD-L1 ISVD-9 interacts with Arg13 of the human PD-L1 N-terminal IgV domain. When Arg13 of the human PD-L1 N-terminal IgV domain was substituted with Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 decreased by approximately 90-fold. The residue Ser100 (SEQ ID NO: 6) of PD-L1 ISVD-9 interacts with Glu 58 of the human PD-L1 N-terminal IgV domain. When Glu 58 of the human PD-L1 N-terminal IgV domain was substituted with Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 decreased by approximately 25-fold. The residue Thr105 (SEQ ID NO: 6) of PD-L1 ISVD-9 interacts with Gln 66 of the human PD-L1 N-terminal IgV domain. When Gln 66 of the human PD-L1 N-terminal IgV domain was substituted with Ala, the binding affinity between PD-L1 ISVD-9 and PD-L1 decreased by approximately 82-fold. Furthermore, the residues D61, N63, V68, M115, S117, Y123, and R125 of the human PD-L1 N-terminal IgV domain may be involved in the interaction between PD-L1 ISVD-9 and human PD-L1. When these residues were substituted with Ala, the binding affinity decreased by approximately 2 to 10-fold. These results are summarized in Table 2 below.

Table 2

[0186] An ISVD specific for PD-L1 can block the binding of PD-L1 to PD1. In some cases, an ISVD specific for PD-L1 can block the binding of PD-L1 to CD80.

[0187] An ISVD specific for PD-L1 can cross-compete with a control anti-PD-L1 antibody for binding to PD-L1.

[0188] The reference anti-PD-L1 antibody may contain a heavy chain CDR3. The heavy chain CDR3 may contain the amino acid sequence set forth in DSFX1X2PTCX3X4X5X6SSGAFQY (SEQ ID NO: 1), X1 where E or G may be; X2 may be D or Y; X3 may be T or P; X4 may be L or G; X5 may be V or P; and X6 may be T or A. In some cases, the reference anti-PD-L1 antibody may contain a heavy chain CDR3 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 9. The reference anti-PD-L1 antibody may also contain a heavy chain CDR1. The heavy chain CDR1 may contain the amino acid sequence set forth in GX1X2X3X4X5RCMA (SEQ ID NO: 2), where X1 may be K or N; X2 may be M or I; X3 may be S or I; X4 may be S or R; X5 may be R or V. For example, the reference anti-PD-L1 antibody may contain a heavy chain CDR1 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 and 7. In some cases, the reference anti-PD-L1 antibody may contain a heavy chain CDR2. The heavy chain CDR2 may contain the amino acid sequence set forth in any one of SEQ ID NOs: 4, 8, and 11. In some cases, the reference anti-PD-L1 antibody is an ISVD specific for PD-L1 such as an anti-PD-L1 VHH. The reference anti-PD-L1 antibody may contain a heavy chain variable domain. The reference anti-PD-L1 antibody may contain a heavy chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15. For example, the heavy chain variable domain may contain the amino acid sequence set forth in SEQ ID NO: 6.

[0189] In the present disclosure, an ISVD specific to PD-L1 (such as those included in the dimers of the present disclosure) may include a heavy chain CDR3. The heavy chain CDR3 may include the amino acid sequence set forth in DSFX1X2PTCX3X4X5X6SSGAFQY (SEQ ID NO: 1), where X1 may be E or G; X2 may be D or Y; X3 may be T or P; X4 may be L or G; X5 may be V or P; and X6 may be T or A. For example, an ISVD specific to PD-L1 may include a heavy chain CDR3 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 9.

[0190] For example, an ISVD specific to PD-L1 may include a heavy chain CDR3 consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5 and 9. In some cases, the heavy chain CDR3 may include an amino acid sequence having 1 or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 5 and 9.

[0191] In the present disclosure, an ISVD specific to PD-L1 (such as those included in the dimers of the present disclosure) may also include a heavy chain CDR1. The heavy chain CDR1 may include the amino acid sequence set forth in GX1X2X3X4X5RCMA (SEQ ID NO: 2), where X1 may be K or N; X2 may be M or I; X3 may be S or I; X4 may be S or R; X5 may be R or V. For example, an ISVD specific to PD-L1 may include a heavy chain CDR1 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 3 and 7.

[0192] For example, an ISVD specific to PD-L1 may include a heavy-chain CDR1 consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3 and 7. In some cases, the heavy-chain CDR1 may include an amino acid sequence having 1 or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 3 and 7.

[0193] In the present disclosure, an ISVD specific to PD-L1 (such as those included in the dimers of the present disclosure) may further include a heavy-chain CDR2. The heavy-chain CDR2 may include any suitable amino acid sequence. In some cases, an ISVD specific to PD-L1 may include a heavy-chain CDR2 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 4, 8, and 11.

[0194] For example, an ISVD specific to PD-L1 may include a heavy chain CDR2 consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 4, 8, and 11. In some cases, the heavy chain CDR2 may include an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 4, 8, and 11.

[0195] In the present disclosure, an ISVD specific to PD-L1 (contained in the dimers of the present disclosure) may include a heavy chain variable domain. The ISVD specific to PD-L1 may include a heavy chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15. For example, the heavy chain variable domain may include the amino acid sequence set forth in SEQ ID NO: 6.

[0196] For example, an ISVD specific to PD-L1 may comprise a heavy chain variable domain consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15. Optionally, an ISVD specific to PD-L1 may comprise a heavy chain variable domain consisting of an amino acid sequence having 1 or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15.

[0197] In the present disclosure, an ISVD specific to PD-L1 may include the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15. For example, an ISVD specific to PD-L1 (included in the dimer of the present disclosure) may include the amino acid sequence set forth in SEQ ID NO: 6. For example, an ISVD specific to PD-L1 may include an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15. In some cases, an ISVD specific to PD-L1 may include an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 6, 10, 12, 13, 14, and 15.

[0198] In some cases, an ISVD specific to PD-L1 may include or be composed of a heavy chain variable domain (VH or VHH).

[0199] For example, an ISVD specific to PD-L1 may be selected from PD-L1 ISVD-9, PD-L1 ISVD-6, PD-L1 ISVD-m3, PD-L1 ISVD-4, PD-L1 ISVD-11, and PD-L1 ISVD-13.

[0200] An ISVD specific for CTLA4 can specifically bind to human CTLA4. For example, an ISVD specific for CTLA4 may be one that can specifically bind to an epitope of the extracellular domain of human CTLA4, and such epitopes may include those described in CN107400166A, Udupi A. Ramagopal, et al., PNAS 2017 May, 114 (21), etc.

[0201] An ISVD specific for CTLA4 can block the binding of CTLA4 to CD80. In some cases, an ISVD specific for CTLA4 can block the binding of CTLA4 to CD86. In some cases, an ISVD specific for CTLA4 can be humanized.

[0202] An ISVD specific for CTLA4 can cross-compete with a reference anti-CTLA4 antibody for binding to CTLA4.

[0203] The reference anti-CTLA4 antibody may contain a heavy chain CDR3. The heavy chain CDR3 may contain the amino acid sequence described in SEQ ID NO: 19. The reference anti-CTLA4 antibody may also contain a heavy chain CDR1. The heavy chain CDR1 may contain the amino acid sequence described in SEQ ID NO: 17. In some cases, the reference anti-CTLA4 antibody may contain a heavy chain CDR2. The heavy chain CDR2 may contain the amino acid sequence described in AIX1X2GGGSTYYADSVKG (SEQ ID NO: 16), where X1 may be Y or S; X2 may be I or L. For example, the heavy chain CDR2 may contain the amino acid sequence described in any one of SEQ ID NOs: 18, 21, and 23. In some cases, the reference anti-CTLA4 antibody is an ISVD specific for CTLA4, such as an anti-CTLA4 VHH. The reference anti-CTLA4 antibody may contain a heavy chain variable domain. The reference anti-CTLA4 antibody may contain a heavy chain variable domain consisting of the amino acid sequence described in any one of SEQ ID NOs: 20, 22, and 24 - 32. For example, the heavy chain variable domain may contain the amino acid sequence described in SEQ ID NO: 20.

[0204] In the present disclosure, an ISVD specific for CTLA4 (such as those included in the dimers of the present disclosure) may include a heavy chain CDR3. The heavy chain CDR3 may include the amino acid sequence set forth in SEQ ID NO: 19.

[0205] In some cases, an ISVD specific for CTLA4 may include a heavy chain CDR3 consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity to the amino acid sequence set forth in SEQ ID NO: 19. In some cases, the heavy chain CDR3 may include an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in SEQ ID NO: 19.

[0206] In the present disclosure, an ISVD specific for CTLA4 (such as those included in the dimers of the present disclosure) may include a heavy chain CDR1. The heavy chain CDR1 may include the amino acid sequence set forth in SEQ ID NO: 17.

[0207] In some cases, the ISVD specific for CTLA4 may include a heavy chain CDR1 consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in SEQ ID NO: 17. In some cases, the heavy chain CDR1 may include an amino acid sequence having one or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in SEQ ID NO: 17.

[0208] In the present disclosure, the ISVD specific for CTLA4 (e.g., as contained in the dimers of the present disclosure) may further include a heavy chain CDR2. The heavy chain CDR2 may include the amino acid sequence set forth in AIX1X2GGGSTYYADSVKG (SEQ ID NO: 16), where X1 may be Y or S; and X2 may be I or L. In some cases, the ISVD specific for CTLA4 may include a heavy chain CDR2 consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 18, 21, and 23.

[0209] For example, an ISVD specific to CTLA4 may include a heavy chain CDR2 consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 18, 21, and 23. In some cases, the heavy chain CDR2 may include an amino acid sequence having 1 or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 18, 21, and 23.

[0210] In the present disclosure, an ISVD specific to CTLA4 (such as that included in the dimers of the present disclosure) may include a heavy chain variable domain. An ISVD specific to CTLA4 may include a heavy chain variable domain consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 20, 22, and 24-32. For example, the heavy chain variable domain may include the amino acid sequence set forth in SEQ ID NO: 20.

[0211] For example, an ISVD specific to CTLA4 may include a heavy chain variable domain consisting of an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 20, 22, and 24-32. In some cases, an ISVD specific to CTLA4 may include a heavy chain variable domain consisting of an amino acid sequence having 1 or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 20, 22, and 24-32.

[0212] In the present disclosure, an ISVD specific to CTLA4 may include the amino acid sequence set forth in any one of SEQ ID NOs: 20, 22, and 24-32. For example, an ISVD specific to CTLA4 (included in the dimer of the present disclosure) may include the amino acid sequence set forth in SEQ ID NO: 20.

[0213] For example, an ISVD specific to CTLA4 may include an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 20, 22, and 24 - 32. In some cases, an ISVD specific to CTLA4 may include an amino acid sequence having 1 or more (e.g., 1 - 2, 1 - 3, 1 - 4, 1 - 5, 1 - 6, 1 - 7, 1 - 8, 1 - 9, 1 - 10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 20, 22, and 24 - 32.

[0214] In some cases, an ISVD specific to CTLA4 may include or be composed of a heavy chain variable domain (VH or VHH).

[0215] For example, an ISVD specific to CTLA4 may be selected from CTLA4 ISVD - 34, CTLA4 ISVD - C1, CTLA4 ISVD - 13, CTLA4 ISVD - 26, CTLA4 ISVD - 27, CTLA4 ISVD - 28, CTLA4 ISVD - 29, CTLA4 ISVD - 30, CTLA4 ISVD - 31, CTLA4 ISVD - 32, and CTLA4 ISVD - 33.

[0216] For example, the dimer of the present application may include or consist of two polypeptide chains. The amino acid sequences of the two polypeptide chains may be the same or different. In some cases, the dimer of the present disclosure may be a homodimer.

[0217] In the present disclosure, one or both of the two polypeptide chains of the dimer may comprise the amino acid sequence according to any one of claims 40 to 43, 46, 48, and 50. For example, one or both of the two polypeptide chains of the dimer may comprise the amino acid sequence set forth in SEQ ID NO: 40.

[0218] In a specific example, one or both of the two polypeptide chains of the dimer may comprise an amino acid sequence having at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) identity with the amino acid sequence set forth in any one of SEQ ID NOs: 40 to 43, 46, 48, and 50. In some cases, one or both of the two polypeptide chains of the dimer may comprise an amino acid sequence having 1 or more (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or more) amino acid deletions, insertions, and / or substitutions in the sequence set forth in any one of SEQ ID NOs: 40-43, 46, 48, and 50.

[0219] As an example, an ISVD specific for PD-L1 may be fused (directly or indirectly, for example via a linker such as a peptide linker) to the N-terminal amino acids of an ISVD specific for CTLA4 to form a bispecific binding moiety. Then, one such bispecific binding moiety may be fused (directly or indirectly, for example via a linker such as a peptide linker) to the N-terminal amino acids of one of the Fc subunits of the present disclosure to provide one polypeptide chain of the dimer. Then, another such bispecific binding moiety may be fused (directly or indirectly, for example via a linker such as a peptide linker) to the N-terminal amino acids of another Fc subunit of the present disclosure to provide the other polypeptide chain of the dimer. The two Fc subunits of the two polypeptide chains may bind to each other (for example, via non-covalent interactions and / or disulfide bonds or other covalent bonds, and in some cases, such covalent bonds are not peptide bonds) to form a dimer. The two biospecific binding moieties may be the same or different. The two Fc subunits may be the same or different.

[0220] In some embodiments, the dimer is a proteinaceous homodimer that includes two identical polypeptide chains, each polypeptide chain includes one of the bispecific binding moieties fused to one of the Fc subunits, and the two Fc subunits bind to each other to form a proteinaceous homodimer. The two Fc subunits may bind to each other via non-covalent interactions and / or disulfide bonds or other covalent bonds, and in some cases, such covalent bonds are not peptide bonds.

[0221] Figures 1A-1B provide an example of a dimer of the present disclosure, where 1 shows an ISVD specific for PD-L1, 2 shows an ISVD specific for CTLA4, 3 shows an Fc domain including an Fc subunit, and 4 shows a bispecific binding moiety.

[0222] The dimers of the present disclosure can compete with CD80 and / or CD86 for binding to CTLA4. For example, competition can be examined in in vitro experiments using a CTLA4-expressing cell line, such as a CTLA4-expressing HEK293 cell line. As another example, competition can be examined in an ELISA assay, such as a competitive ELISA assay.

[0223] The dimers of the present disclosure can compete with PD1 and / or CD80 for binding to PD-L1. For example, competition can be examined in in vitro experiments using a PD-L1-expressing cell line, such as a PD-L1-expressing A375 cell line. As another example, competition can be examined in an ELISA assay, such as a competitive ELISA assay.

[0224] The dimers of the present disclosure can block the binding of PD-L1 to PD-1. In some cases, the dimers of the present disclosure can block the binding of PD-L1 to CD80. In some cases, the dimers of the present disclosure can block the binding of CTLA4 to CD80. In some cases, the dimers of the present disclosure can block the binding of CTLA4 to CD86.

[0225] The dimers of the present disclosure can bind to CTLA4 with a K of about 1×10 -6 M or less, for example, about 1×10 -7 M or less, about 1×10 -8 M or less, about 0.5×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, or a value therebelow. D

[0226] The dimers of the present disclosure can bind to PD-L1 with a K of about 1×10 -6 M or less, for example, about 1×10 -7 M or less, about 1×10 -8 M or less, about 0.5×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, or a value therebelow. D ​

[0227] The dimers of the present disclosure can stimulate the secretion of immunomodulatory substances (such as IL-2) by immune cells (such as PBMC cells).

[0228] For example, the dimers of the present disclosure can be selected from aPDL1.9-aCTLA4.34-Fc, aPDL1.9-L-aCTLA4.34-Fc, aCTLA4.34-aPDL1.9-Fc, aCTLA4.34-L-aPDL1.9-Fc, aPDL1.6-aCTLA4.34-Fc, aPDL1.m3-aCTLA4.34-Fc and aPDL1.9-aCTLA4.13-Fc.

[0229] For example, the dimers of the present disclosure may contain an ISVD specific for CTLA4 and an ISVD specific for PDL1. The ISVD specific for PD-L1 may include a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 5, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 4, and a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 3. Also, the ISVD specific for CTLA4 may include a CDR3 containing the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 containing the amino acid sequence set forth in SEQ ID NO: 18, and a CDR1 containing the amino acid sequence set forth in SEQ ID NO: 17. Further, the dimers of the present disclosure may contain an ISVD specific for PD-L1 containing the amino acid sequence set forth in SEQ ID NO: 6 and an ISVD specific for CLTA4 containing the amino acid sequence set forth in SEQ ID NO: 20. For example, the dimers of the present disclosure may contain the amino acid sequence of SEQ ID NO: 40.

[0230] In addition, the dimers of the present disclosure can be named KN046.

[0231] Her2 inhibitor

[0232] In the present disclosure, the Her2 inhibitor can be a Her2 antibody or its antigen-binding portion and / or its complex.

[0233] In some embodiments, the Her2 inhibitor can be a bispecific antibody or an antigen-binding portion thereof, the bispecific antibody or an antigen-binding portion thereof can have a common light chain, and the common light chain refers to two light chains having the same sequence.

[0234] In some embodiments, the heavy chains of the bispecific antibody or an antigen-binding portion thereof can be capable of correctly associating with the respective light chains under physiological conditions or during in vitro protein expression.

[0235] The common light chain may be designed from those in which at least one of the light chain sequences of two original monoclonal antibodies (known monoclonal antibodies) is different. In some embodiments, the common light chain may be identical to one of the two original monoclonal antibodies or may be modified based on one of them (e.g., amino acid sequence modification), and the purpose of the modification is to maintain the affinity for each antigen or epitope. In some embodiments, it may include amino acid sequence modification, amino acid mutation, deletion or addition, for example, mutation, deletion or addition of 3 or fewer amino acids, preferably 2 or fewer amino acids, more preferably 1 or fewer amino acids.

[0236] In this application, the light chain sequences (especially variable region sequences) of two monoclonal antibodies (i.e., original antibodies) against different antigens or antigen epitopes are analyzed and verified, and a common light chain capable of associating with the heavy chains of the two monoclonal antibodies can be obtained. Even after binding to the heavy chain, the common light chain can specifically bind to the antigen or antigen epitope targeted by the original monoclonal antibody.

[0237] In this application, the light chain sequences (especially variable region sequences) of two monoclonal antibodies (i.e., original antibodies) against different antigens or antigen epitopes are analyzed and verified, and a common light chain capable of associating with the heavy chains of the two monoclonal antibodies can be obtained. Even after binding to the heavy chain, the common light chain can specifically bind to the antigen or antigen epitope targeted by the original monoclonal antibody.

[0238] In the present application, the common light chain can be used to express a bispecific antibody and can also be used to express a mixture containing two antibodies. When a bispecific antibody is expressed, the antibody includes a light chain and a heavy chain that can bind to a first antigen, and a light chain and a heavy chain that can bind to a second antigen, and the sequences of the two light chains are exactly the same, that is, a common light chain.

[0239] In the present application, the light chain constant regions of the two original antibodies can be of the κ type or the λ type; the κ type light chain constant region includes various allotypes such as Km1, Km2, and Km3; the λ type light chain constant region includes various allotypes such as CL1, CL2, CL3, CL6, and CL7.

[0240] It is known in the art that the variable region can be important for the specific binding between an antigen and an antibody. Therefore, during the process of modifying or obtaining an antibody, the selection and modification of the variable region sequence are important. Therefore, in the present application, in order to obtain a bispecific antibody or an antibody mixture having a common light chain, first, it is necessary to obtain the variable region of the common light chain. According to the method described above, after selecting the light chain variable region of one original monoclonal antibody or its variant as the variable region of the common light chain, the constant region of the common light chain is determined. Usually, the common light chain constant region can be determined to be the light chain constant region of the monoclonal antibody from which the common light chain variable region is derived. In some cases, the light chain constant region of another monoclonal antibody can be determined as the common light chain constant region. If necessary, in order to obtain a more suitable common light chain constant region, based on the knowledge known in the art, the original light chain constant region can be modified (for example, amino acid addition, deletion, or mutation, etc.). For example, the modified common light chain constant region has characteristics such as better ADCC, CDC, endocytosis, stability, immunogenicity, or half-life.

[0241] In this application, the types of the heavy chains of the two original antibodies may be the same or different, and preferably, the types of the heavy chains are the same. In one embodiment, when preparing a bispecific antibody and an antibody mixture, the sequences of the variable region of the heavy chain and the CH1 domain are not changed as compared with the sequences of the original antibodies.

[0242] In this application, the two arms of the bispecific antibody or the antibodies in the mixture containing two antibodies may all be derived from two original monoclonal antibodies. When preparing a bispecific antibody or an antibody mixture, only the sequence of the variable region of the light chain is changed to obtain a common light chain, but the sequence of the variable region of the heavy chain may not need to be changed. That is, in the prepared bispecific antibody or antibody mixture, the sequence of the antibody heavy chain variable region may be the same as the sequence of the original antibody, but the sequence of at least one light chain variable region must be different from the sequence of the original antibody.

[0243] In this application, the two original monoclonal antibodies may be selected according to different requirements or purposes. For example, the two selected monoclonal antibodies may be against different antigen epitopes of the same antigen. Alternatively, one of the selected antibodies binds to a related antigen on the surface of tumor cells, and the other antibody can trigger immune effector cells to further kill the cells.

[0244] In this application, when preparing a bispecific antibody, in order to promote the formation of heterodimeric proteins during antibody expression, the heavy chain (such as the Fc fragment) can be modified based on techniques known in the art.

[0245] In this application, when preparing an antibody mixture, the heavy chain (such as the Fc fragment) can be modified based on techniques known in the art to promote the formation of homodimeric proteins during antibody expression.

[0246] In the present application, techniques for modifying the Fc fragment of an antibody heavy chain to promote the formation of homodimeric proteins or heterodimeric proteins are known in the art, and for example, Ridgway, Presta et al. 1996, Carter 2001, Patent CN102558355A, and Patent CN103388013A may be mentioned.

[0247] In the present application, as a technique for fusing polypeptides that recognize different antigen epitopes, for example, heterodimeric Fc fusion techniques as shown in the examples can be considered, but are not limited thereto, and "Fab" techniques may also be used. See Figure 2.

[0248] In the present application, the heterodimeric Fc fusion technique used may be based on the "knob"-"hole" model or the "charge repulsion model", but is not limited to these two models.

[0249] In some embodiments, the heavy chain Fc segment of a bispecific antibody or its antigen-binding portion may be designed to be more favorable for the formation of heterodimeric proteins.

[0250] In some embodiments, the two original monoclonal antibodies may be pertuzumab and trastuzumab.

[0251] In some embodiments, a common light chain can associate with the heavy chains of pertuzumab and trastuzumab, respectively.

[0252] In some embodiments, the common light chain can be selected from the light chain of pertuzumab or trastuzumab or a mutant thereof. In some embodiments, the heavy chain (including the variable region and the constant region) of a bispecific antibody or its antigen-binding portion can be the same as the two original monoclonal antibodies or can be modified to facilitate formation. Heterodimeric protein; for example, modify the heavy chain Fc segment to promote the formation of heterodimeric proteins.

[0253] In some embodiments, the common light chain can be selected from the light chain of pertuzumab or trastuzumab or mutants thereof. In some embodiments, the heavy chain (including the variable region and the constant region) of the bispecific antibody or its antigen-binding portion can be identical to the two original monoclonal antibodies or can be modified to facilitate formation. Heterodimeric protein; for example, to promote the formation of the heterodimeric protein, the heavy chain Fc segment is modified.

[0254] In some embodiments, the sequence of the variable region of the common light chain can include a sequence selected from amino acids 1 to 107 of SEQ ID NOs: 65-70. In some embodiments, the sequence of the light chain constant region can include the sequence of amino acids 108 to 214 of SEQ ID NO: 65. For example, the variable region of the common light chain of the Her2 inhibitor may be the variable region of the light chain of trastuzumab.

[0255] In some embodiments, the variable regions of the heavy chains of the antibody or its antigen-binding portion can be the variable region of the heavy chain of pertuzumab and the variable region of the heavy chain of trastuzumab, respectively; for example, the variable regions of the heavy chains may each include the sequences set forth in SEQ ID NO: 87 and SEQ ID NO: 88.

[0256] In some embodiments, the sequences of the variable regions of the two heavy chains can each include the sequences set forth in SEQ ID NOs: 89 and 90.

[0257] In one embodiment, the two heavy chains can each include the sequences set forth in SEQ ID NOs: 83 and 84.

[0258] For example, the common light chain of the Her2 inhibitor can include the amino acid sequence set forth in SEQ ID NO: 65. The variable regions of the heavy chains can each include the sequences set forth in SEQ ID NOs: 87 and 88, and the variable regions of the two heavy chains can each include the amino acid sequences set forth in SEQ ID NOs: 89 and 90. The two heavy chains can each include the sequences set forth in SEQ ID NOs: 83 and 84.

[0259] Also, the Her2 inhibitor of the present disclosure can be named KN026.

[0260] Pharmaceutical Composition and Use

[0261] The present disclosure provides a pharmaceutical composition comprising an effective amount of the immune checkpoint inhibitor and an effective amount of the Her2 inhibitor of the present disclosure, and optionally a pharmaceutically acceptable excipient.

[0262] In some embodiments, the term "effective amount" refers to the amount of the immune checkpoint inhibitor or Her2 inhibitor of the present disclosure that is effective to at least partially alleviate, suppress, prevent, and / or ameliorate a condition, or disorder or disease when administered to a subject. The effective amount of the pharmaceutical composition of the present invention can be administered for the prevention or treatment of a disease. The appropriate dosage of the pharmaceutical composition can be determined based on the type of disease to be treated, the type of pharmaceutical composition, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0263] In another form, the present disclosure provides the use of the pharmaceutical composition of the present disclosure in the preparation of a medicament for the treatment of tumors in a subject.

[0264] In another form, the present disclosure provides the pharmaceutical composition of the present disclosure for use in the treatment of tumors in a subject.

[0265] In another form, the present disclosure provides a method for treating tumors in a subject, comprising the pharmaceutical composition of the present disclosure.

[0266] In some embodiments, the treatment results in a sustained response in the individual after the treatment is discontinued. The medicaments described herein can find use in the treatment of conditions where an improvement in immunogenicity is desired, such as an improvement in tumor immunogenicity for the treatment of tumors. Further, the pharmaceutical composition can enhance the immune function in an individual having a tumor by administering an effective amount of the immune checkpoint and Her2 inhibitor to the individual.

[0267] In the present disclosure, the tumor may be selected from the group consisting of solid tumors and hematological tumors.

[0268] In the present disclosure, the tumor may be selected from the group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, cervical cancer, ovarian cancer, endometrial cancer, biliary tract cancer, colorectal cancer, and urothelial cancer.

[0269] In the present disclosure, the tumor may be selected from the group consisting of Her2-abnormal solid tumors and Her2-positive cancers.

[0270] In the present disclosure, the subject may be administered an anti-Her2 antibody and / or an anti-PD1 agent.

[0271] For example, the anti-Her2 antibody may include trastuzumab.

[0272] For example, the subject may be administered chemotherapy.

[0273] In some embodiments, the chemotherapy may include primary chemotherapy and / or secondary chemotherapy. For example, the secondary chemotherapy may include monotherapy with paclitaxel + ramucirumab, paclitaxel, docetaxel, and / or irinotecan.

[0274] In the present disclosure, the chemotherapy may refer to any treatment of the tumor with a chemical agent. The chemical agent can kill tumor cells, shrink the tumor, and / or relieve cancer signs and symptoms. For example, the chemotherapy may include primary chemotherapy and / or secondary chemotherapy.

[0275] In the present disclosure, the primary chemotherapy may refer to a chemotherapy regimen or regimen generally accepted for the initial treatment of a cancer of a given type and stage. For example, the primary chemotherapy may include platinum-based chemotherapy. In some embodiments, the primary platinum-based chemotherapy may include chemotherapy with a platinum (P) compound (cisplatin or carboplatin).

[0276] In the present disclosure, the secondary chemotherapy may refer to that which is attempted when the primary selected chemotherapy does not function sufficiently. For example, the secondary chemotherapy may include monotherapy with paclitaxel + ramucirumab, paclitaxel, docetaxel, and / or irinotecan.

[0277] In the present disclosure, the Her2-positive cancer may refer to a cancer having tumor cells with a Her2 expression level higher than the normal value.

[0278] In the present disclosure, the Her2-abnormal solid tumor may refer to a solid tumor having Her2 abnormality. For example, the solid tumor may have abnormal Her2 gene amplification, Her2 gene mutation, and overexpression of Her2 protein.

[0279] In the present disclosure, the anti-PD1 agent may refer to any agent that binds to PD1 and / or blocks the binding between PD1 and PD-L1. For example, the anti-PD1 agent may include an anti-PD1 antibody.

[0280] In the present disclosure, the tumor may include the Her2-abnormal solid tumor in which the subject has failed previous trastuzumab therapy.

[0281] In the present disclosure, the tumor may include Her2-positive GC (gastric cancer) and Her2-positive GEJ (gastroesophageal junction adenocarcinoma) in which the subject has failed at least one previous treatment of systemic therapy (for example, failed previous trastuzumab therapy, or failed both trastuzumab and anti-PD-1 agent therapy).

[0282] The pharmaceutical composition of the present invention may be administered by the same administration route or different administration routes. In some embodiments, the pharmaceutical composition may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the Her2 inhibitor may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the immune checkpoint inhibitor may be administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[0283] In the present disclosure, the Her2 inhibitor may be administered by intravenous administration.

[0284] In the present disclosure, the immune checkpoint inhibitor may be administered by intravenous administration.

[0285] As a general proposition, the effective amount of the immune checkpoint inhibitor and the effective amount of the Her2 inhibitor administered to a subject may be in the range of about 0.01 to about 100 mg / kg of the subject's body weight, regardless of whether it is by one or more administrations.

[0286] In some embodiments, the Her2 inhibitor may be administered at about 0.01 to about 100 mg / kg, about 1 mg / kg to about 80 mg / kg, about 1 mg / kg to about 70 mg / kg, about 1 mg / kg to about 60 mg / kg, about 1 mg / kg to about 50 mg / kg, about 1 mg / kg to about 40 mg / kg, about 1 mg / kg to about 30 mg / kg, at about 1 mg / kg to 100 mg / kg administration, at about 5 mg / kg to about 100 mg / kg administration, at about 10 mg / kg to about 100 mg / kg administration, or at about 20 mg / kg to about 100 mg / kg. In some embodiments, the Her2 inhibitor may be administered at about 20 to about 30 mg / kg.

[0287] In some embodiments, the immune checkpoint inhibitor can be administered at about 0.01 to about 100 mg / kg, about 0.01 mg / kg to about 80 mg / kg, about 0.01 mg / kg to about 60 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 4 mg / kg, or about 0.01 mg / kg to about 3 mg / kg. In some embodiments, the immune checkpoint inhibitor may be administered at a dose of about 1 to about 5 mg / kg. For example, the immune checkpoint inhibitor may be administered at a dose of about 3 mg / kg to about 5 mg / kg.

[0288] For example, the immune checkpoint inhibitor may be administered at a dose of about 3 mg / kg, and the Her2 inhibitor may be administered at a dose of about 20 mg / kg or about 30 mg / kg. For example, the immune checkpoint inhibitor may be administered at a dose of about 5 mg / kg, and the Her2 inhibitor may be administered at a dose of about 20 mg / kg.

[0289] Administration may be a single administration or multiple administrations (e.g., 2 or 3 administrations) such as infusion. The dose of the antibody administered in combination therapy can be reduced compared to monotherapy. The course of this treatment method can be easily observed by conventional techniques.

[0290] The pharmaceutical composition can be administered at about 0.01 to about 100 mg / kg, about 0.01 mg / kg to about 80 mg / kg, about 0.01 mg / kg to about 60 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, or about 0.01 mg / kg to about 3 mg / kg.

[0291] The pharmaceutical composition may be administered to the subject at a dosing frequency of once every four weeks, once every two weeks, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or at a frequency lower than that, as long as a therapeutic response is obtained. In certain embodiments, the Her2 inhibitor may be administered to the subject at a dosing frequency of once every four weeks, once every two weeks, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every eight weeks, once every twelve weeks, or at a frequency lower than that, as long as a therapeutic response is obtained. In certain embodiments, the immune checkpoint may be administered to the subject at a dosing frequency of about 4 times / week, 2 times / week, 1 time / week, 2 weeks / week, 3 weeks / week, 4 weeks / week, 5 weeks / week, 6 weeks / week, 8 weeks / week, 9 weeks / week, 12 weeks / week, or at a frequency lower than that, as long as a therapeutic response is obtained.

[0292] In the present disclosure, the Her2 inhibitor may be administered once every two weeks or once every three weeks, or as a loading dose. In the present disclosure, the immune checkpoint inhibitor may be administered once every two weeks or once every three weeks.

[0293] For example, the immune checkpoint inhibitor may be administered once every two weeks, and the Her2 inhibitor may be administered once every two weeks. For example, the immune checkpoint inhibitor may be administered once every three weeks, and the Her2 inhibitor may be administered once every two weeks. For example, the immune checkpoint inhibitor may be administered once every three weeks, and the Her2 inhibitor may be administered once every three weeks.

[0294] The present disclosure provides for the use of an immune checkpoint inhibitor in combination with a Her2 inhibitor in the preparation of a medicament for treating tumors in a subject in need thereof. The use of the term "in combination with" does not limit the order in which the treatments are administered to a subject in need thereof. The immune checkpoint inhibitor is administered to a subject in need of a Her2 inhibitor before (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly, or simultaneously, or after (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after), simultaneously or subsequently.

[0295] Examples

[0296] The following examples are set forth to provide a complete disclosure and description of the method of manufacture and method of use of the invention to those skilled in the art and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to indicate that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numerical values (e.g., amounts, temperatures, etc.) used, but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations may be used, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; nt, nucleotide; i.m, intramuscular; i.p, intraperitoneal; s.c, subcutaneous, etc.

[0297] Example 1 Clinical study of a Her2 inhibitor in combination with the dimer of the present disclosure in subjects with Her2-positive tumors

[0298] This trial is an open-label, Phase 1b, multi-center clinical trial. The purpose of this trial is to evaluate the safety and efficacy of the combination of a Her2 inhibitor and the dimer of the present disclosure in subjects with Her2-positive tumors.

[0299] This trial includes a dose-escalation period and a dose-expansion period. The dose escalation of the Her2 inhibitor is 20 mg / kg Q2W and 30 mg / kg Q2W, and the dose escalation of the dimer is 3 mg / kg Q2W. In this phase, mTPI (Modified Toxicity Probability Interval) is implemented. mTPI-2 determines the maximum tolerated dose (MTD) according to the calculated design while increasing or decreasing the dose. Apply the isotonic regression of mTPI-2 to the observed DLT (dose-limiting toxicity), and estimate the MTD as the dose closest to the target DLT after the dose-escalation phase.

[0300] The first treatment group (Her2 inhibitor 6 20 mg / kg Q2W + dimer 3 mg / kg Q2W) will have 3 to 6 subjects. SMC is performed after DLT observation. Subjects in the first treatment group will be classified into the second treatment group (Her2 inhibitor 6 30 mg / kg Q2W + dimer 3 mg / kg Q2W) if they meet the "ascending to high dose" criterion according to the mTPI-2 design table. The second group targets 3 to 6 subjects, and the first treatment group is expanded to 12 subjects. When the subjects meet the "transition to high dose" or "maintain the original dose" criterion and all DLT observations in the dose-escalation period are completed, the second dose group is expanded to 12 subjects.

[0301] After the dose-escalation phase, the SMC determines the RP2D (Recommended Phase II Dose), and this trial enters the dose-expansion phase. The SMC determines the original dose, the intermediate dose-increase group, or other increase groups (e.g., different dosing intervals such as fixed-dose administration, Q3W) at the same dose level according to the safety, PK data, and / or other data of the Her2 inhibitor and the dimer, or conducts a high-dose trial (e.g., dimer 5 mg / kg). Primary evaluation items of the trial: Dose-escalation period: DLT (dose-limiting toxicity); Expansion phase: ORR (Objective Response Rate) and DOR (Duration of Response) determined by RECIST 1.1. This result indicates that the combination of a Her2 inhibitor and the dimer of the present disclosure can effectively treat the Her2-positive tumors of the subjects.

[0302] Example 2 Preliminary evaluation results of the safety, tolerability and efficacy in the combined administration of KN026 (Her2-targeted bispecific antibody) and KN046 (anti-PD-L1 / CTLA-4 bispecific antibody) in patients with Her2 ectopic solid cancer

[0303] Background:

[0304] Since Her2 strongly suppresses innate immunity via the cGAS-STING signal, it is expected that ADCP by Her2 antibody will also lead to immunosuppression mediated by macrophages. Both preclinical and clinical trials have suggested that the combination of anti-Her2 antibody and immune checkpoint inhibitor enables the coordinated action of innate immunity and adaptive immunity. KN026 is a novel bispecific antibody that simultaneously binds to two different Her2 epitopes. KN046 is a novel bispecific antibody that inhibits both the interaction between PD-L1 and PD-1 / CD80 and the interaction between CTLA-4 and CD80 / CD86. Herein, the interim results of the Phase Ib dose-escalation study and expansion study evaluating the safety, tolerability and efficacy of the combination of KN026 and KN046 in patients with solid cancer with Her2 abnormality are reported.

[0305] Test method:

[0306] This trial was conducted on patients with locally confirmed Her2 abnormalities (Her2 mutations, Her2 amplifications, Her2 overexpressions) who were refractory to standard treatment. The subjects were administered KN026 and KN046 in three dose levels in combination until disease progression, unacceptable toxicity, or withdrawal of consent (DL1). KN026 20 mg / kg Q2W + KN046 3 mg / kg Q2W; DL2: KN026 20mg / kg Q2W, loading dose on days 1 and 8 of the first cycle + KN046 5mg / kg Q3W; DL3: KN026 30mg / kg Q3W, loading dose on days 1 and 8 of the first cycle + KN046 5mg / kg Q3W. Tumor responses were evaluated Q8W based on RECIST 1.1. The primary evaluation item was DLT, and the main secondary evaluation items were efficacy parameters (ORR, DOR, PFS).

[0307] Trial results:

[0308] As of September 8, 2020, 25 cases were enrolled in DL1 (n = 20, dose escalation 3), DL2 (n = 3), and DL3 (n = 2) (15 cases of mGC / GEJ, 8 cases of mCRC, and 2 cases of other solid cancers). 15 patients continued treatment, and 10 patients discontinued treatment due to disease progression (5 patients), death (2 patients), and other reasons (3 patients). There were 18 cases of Her2 positivity (12 out of 18 cases had ineffective pretreatment with trastuzumab), 2 cases of Her2 mutation, and 5 cases of Her2 low expression (without FISH amplification). No DLT was observed. Also, no decrease in LVEF or clinically significant cardiac events were observed. Treatment-related adverse events occurred in 23 patients (92%), and among them, treatment-related adverse events of grade 3 or higher occurred in 6 patients (24%). irAE occurred in 11 patients (44%), and most were grade 1 or 2, except for 1 patient who experienced grade 3 immune-mediated endocrine disorder. The main adverse events related to KN026 and KN046 (frequency ≥ 15%) were infusion-related reactions (n = 11, 44.0%), anemia (n = 9, 36.0%), leukopenia (n = 6, 24.0%), diarrhea (n = 5, 20.0%), increased AST (n = 5, 20.0%), thrombocytopenia (n = 5, 20.0%), rash (n = 5, 20.0%), increased ALT (n = 4, 16.0%), etc. The objective response rate of Her2-positive patients (n = 14) was 9 / 14 (64.3%), and the disease control rate was 13 / 14 (92.9%, 95% CI 66.1 - 99.8%). 4 out of 5 cases of Her2 mutation or low expression achieved SD, and among them, 1 case achieved SD for more than 24 weeks. There were 2 deaths due to disease progression, and both had only received 1 cycle of KN026 + KN046 due to COVID-19 restrictions.

[0309] Conclusion: The combined therapy of KN026 and KN046 has high tolerance and preliminary anti-tumor activity was confirmed in Her2-positive solid cancers.

[0310] Clinical trial information: NCT04040699

[0311] Example 3 Advice on Breakthrough Therapy Designation Request (BTDR)

[0312] This book is used as a basis for stating opinions on whether the application for breakthrough therapy designation (BTD) by this department is appropriate, whether it may be too preliminary at present, or whether it may not meet the BTD criteria at present.

[0313] 1. Provide information related to whether the indication is severe and life-threatening. Briefly explain the indication and the disease targeted by the product:

[0314] The indication of this drug is Her2-positive (Her2 IHC3+ or Her2 IHC2+ / FISH+) gastric / gastroesophageal junction adenocarcinoma and esophageal adenocarcinoma (GC / GEJ / EAC).

[0315] 2. Explain the drug overview, mechanism of action (if known), and relevance to existing therapies:

[0316] KN026 is a Her2 bispecific antibody that targets both domains II and IV of Her2, and KN046 is a PD-L1 / CTLA-4 bispecific antibody that blocks the PD1 / PD-L1 and CTLA-4 pathways.

[0317] 3. Briefly explain available therapies if any:

[0318] Regimens based on fluoropyrimidine, platinum agents, and trastuzumab are commonly used as first-line treatment. As second-line treatment, paclitaxel + ramucirumab, paclitaxel, docetaxel, single-agent therapy with irinotecan, and best supportive care are available, and an objective response rate of about 15 - 25% has been obtained. The median overall survival is about 8 - 9 months for second-line treatment and 4 - 6 months for late-stage treatment, and there is still a large medical need.

[0319] 4. Provide information related to preliminary clinical evidence*, such as study design, study endpoints, treatment groups, number of enrolled subjects, etc.:

[0320] KN046-IST-02 (NCT04040699) is an ongoing dose-escalation and expansion study to evaluate the efficacy and safety of KN046 and KN026 in patients with Her2-positive solid cancers. As of September 3, 2020, 14 patients with Her2-positive solid cancers (including 9 Her2-positive GC / GEJ patients) have been enrolled and their efficacy is being evaluated. An efficacy rate of 64.3% (9 / 14) was confirmed in Her2-positive solid cancers and 66.7% (6 / 9) in Her2-positive GC / GEJ. The 14 patients had no prior response to trastuzumab, and an objective response rate of 60% (6 / 10) was observed. Two patients had no response to both trastuzumab and anti-PD-1 drugs, with 1 partial response and 1 disease stability observed.

[0321] For example, for products in the oncology / hematology field, preliminary clinical evidence includes response rate, duration of response, degree of prior treatment, etc.

[0322] In this specification, preferred embodiments of the present invention have been shown and described, but it will be apparent to those skilled in the art that such embodiments are provided only by way of example. The present invention is not intended to be limited by the specific examples provided in the specification. Although the present invention has been described with reference to the above specification, the description and illustration of the embodiments in this specification are not intended to be construed in a limiting sense. Without departing from the present invention, numerous variations, modifications, and substitutions can occur to those skilled in the art. Furthermore, it is to be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative ratios described herein that depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein can be used in the practice of the present invention. Therefore, the present invention is considered to include such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that the methods and structures within these claims and their equivalents be covered thereby.

Claims

1. A combination comprising an immune checkpoint inhibitor and a Her2 inhibitor, wherein the combination is used for treating a tumor of a subject in need thereof, and the immune checkpoint inhibitor can specifically bind to PD-L1 and CTLA4. Here, the immune checkpoint inhibitor is a dimer, the dimer is formed by two polypeptide chains, each of the two polypeptide chains contains an antibody Fc subunit, the dimer contains two immunoglobulin single variable domains (ISVDs), one of the ISVDs is specific for PD-L1, and one of the ISVDs is specific for CTLA4. The ISVD specific for CTLA4 contains a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO:

20. The ISVD specific for PD-L1 contains a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO:

6. For both of the two polypeptide chains, the C-terminus of the ISVD specific for PD-L1 is optionally fused via a linker to the N-terminus of the ISVD specific for CTLA4, and the C-terminus of the ISVD specific for CTLA4 is optionally fused via a linker to the N-terminus of the antibody Fc subunit. The Her2 inhibitor is a bispecific antibody, the bispecific antibody has a common light chain and two heavy chains, the sequence of the variable region of the common light chain contains the sequence set forth at amino acid positions 1 to 107 of SEQ ID NO: 65, and the heavy chain variable regions each contain the sequences set forth in SEQ ID NOs: 87 and 88. The tumor is selected from the group consisting of Her2-abnormal solid tumors and Her2-positive cancers.

2. The combination according to claim 1, wherein the antibody Fc subunit is derived from an IgG Fc subunit.

3. The combination according to claim 2, wherein the IgG is human IgG1.

4. The combination according to any one of claims 1 to 3, wherein the antibody Fc subunit contains the amino acid sequence set forth in any one of SEQ ID NOs: 35, 38 and 39.

5. The combination according to any one of claims 1 to 4, wherein the dimer is a homodimer.

6. The combination according to any one of claims 1 to 5, wherein both of the two polypeptide chains contain the amino acid sequence set forth in SEQ ID NO:

40.

7. The combination according to any one of claims 1 to 6, wherein the two heavy chains of the Her2 inhibitor each contain the sequences set forth in SEQ ID NOs: 83 and 84.

8. The combination according to any one of claims 1 to 7, wherein the Her2 inhibitor is administered at a dose of 0.01 mg / kg to 100 mg / kg.

9. The combination according to any one of claims 1 to 8, wherein the Her2 inhibitor is administered at a dose of 20 mg / kg to 30 mg / kg.

10. The combination according to any one of claims 1 to 9, wherein the immune checkpoint inhibitor is administered at a dose of 0.01 mg / kg to 100 mg / kg.

11. The combination according to any one of claims 1 to 10, wherein the immune checkpoint inhibitor is administered at a dose of 3 mg / kg to 5 mg / kg.

12. The combination according to any one of claims 1 to 11, wherein the Her2 inhibitor is administered at an administration frequency of 4 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, or once every 12 weeks.

13. The combination according to any one of claims 1 to 12, wherein the Her2 inhibitor is administered once every 2 weeks or once every 3 weeks, or as a loading dose.

14. The combination according to any one of claims 1 to 13, wherein the immune checkpoint inhibitor is administered at an administration frequency of 4 times a week, 2 times a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, once every 9 weeks, or once every 12 weeks.

15. The combination according to any one of claims 1 to 14, wherein the immune checkpoint inhibitor is administered once every 2 weeks or once every 3 weeks.

16. The combination according to any one of claims 1 to 15, wherein the Her2 inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

17. The combination according to any one of claims 1 to 16, wherein the Her2 inhibitor is administered by intravenous administration.

18. The combination according to any one of claims 1 to 17, which is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

19. The combination according to any one of claims 1 to 18, wherein the immune checkpoint inhibitor is administered by intravenous administration.

20. The combination according to any one of claims 1 to 19, wherein the tumor is selected from the group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, cervical cancer, ovarian cancer, endometrial cancer, biliary tract cancer, colorectal cancer, and urothelial cancer.

21. The combination according to any one of claims 1 to 20, wherein the subject has been administered an anti-Her2 antibody and / or an anti-PD1 agent.

22. The combination according to claim 21, wherein the anti-Her2 antibody comprises trastuzumab.

23. The combination according to any one of claims 1 to 22, wherein the subject is administered chemotherapy.

24. The combination according to claim 23, wherein the chemotherapy comprises primary chemotherapy and / or secondary chemotherapy.

25. The combination according to claim 24, wherein the secondary chemotherapy comprises monotherapy with paclitaxel + ramucirumab, paclitaxel, docetaxel, and / or irinotecan.

26. A pharmaceutical composition for use in treating a tumor, comprising an effective amount of an immune checkpoint inhibitor, an effective amount of a Her2 inhibitor, and optionally a pharmaceutically acceptable excipient. Here, the immune checkpoint inhibitor is a dimer, the dimer is formed by two polypeptide chains, each of the two polypeptide chains contains an antibody Fc subunit, the dimer contains two immunoglobulin single variable domains (ISVDs), one of the ISVDs is specific for PD-L1, one of the ISVDs is specific for CTLA4, the ISVD specific for CTLA4 comprises a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO:

20. the ISVD specific for PD-L1 comprises a heavy chain variable domain consisting of the amino acid sequence set forth in SEQ ID NO:

6. For both of the two polypeptide chains, the C-terminus of the ISVD specific for PD-L1 is optionally fused via a linker to the N-terminus of the ISVD specific for CTLA4, and the C-terminus of the ISVD specific for CTLA4 is optionally fused via a linker to the N-terminus of the antibody Fc subunit. The Her2 inhibitor is a bispecific antibody, the bispecific antibody has a common light chain and two heavy chains, the sequence of the variable region of the common light chain includes the sequence described at amino acid positions 1 to 107 of SEQ ID NO: 65, and the heavy chain variable regions each include the sequences described in SEQ ID NOs: 87 and 88. The tumor is selected from the group consisting of Her2-abnormal solid tumors and Her2-positive cancers.

27. The pharmaceutical composition according to claim 26, wherein the tumor is selected from the group consisting of NSCLC, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, esophageal adenocarcinoma, cervical cancer, ovarian cancer, endometrial cancer, biliary tract cancer, colorectal cancer, and urothelial cancer.

28. The pharmaceutical composition according to claim 27, which is administered at an administration frequency of once every 4 weeks, once every 2 weeks, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 8 weeks, or once every 12 weeks.

29. The pharmaceutical composition according to any one of claims 26 to 28, which is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, by inhalation, intrathecally, intraventricularly, or intranasally.

30. The pharmaceutical composition according to any one of claims 26 to 29, which is administered at a dose of 0.01 mg / kg to 100 mg / kg.

31. The pharmaceutical composition according to any one of claims 27 to 30, wherein the subject suffering from the tumor has been administered an anti-Her2 antibody and / or an anti-PD1 agent.

32. The pharmaceutical composition according to claim 31, wherein the anti-Her2 antibody includes trastuzumab.

33. The pharmaceutical composition according to any one of claims 31 to 32, wherein the subject is administered chemotherapy.

34. The pharmaceutical composition according to claim 33, wherein the chemotherapy includes primary chemotherapy and / or secondary chemotherapy.

35. The pharmaceutical composition according to claim 34, wherein the secondary chemotherapy includes monotherapy with paclitaxel + ramucirumab, paclitaxel, docetaxel, and / or irinotecan.

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