Anti-tigit x pvrig antibodies and uses thereof

Abstract

Provided in the present disclosure are anti-TIGIT / PVRIG antibodies and polypeptide complexes, nucleic acid molecules encoding the same, expression vectors and host cells for expressing the antibodies. Further provided are methods for preventing and treating cancer and immune disorders by administering an anti-TIGIT / PVRIG antibody.

Description

[0001] Cross-referencing

[0002] This application claims the benefit of international patent application PCT / CN2023 / 098602, filed on June 6, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list, which is incorporated herein by reference in its entirety. Technical Field

[0005] This application generally relates to antibodies. More specifically, this application relates to antibody-peptide complexes targeting TIGIT and PVRIG, methods for preparing the antibody-peptide complexes, and uses of the antibodies. Background Technology

[0006] In the tumor microenvironment, persistent antigen stimulation can lead to T cell exhaustion, T cell dysfunction, and overexpression of co-inhibitory receptors including PD-1, LAG-3, TIM3, and TIGIT. Currently, various strategies are being explored to revitalize exhausted T cells using single or combined small molecule or therapeutic antibody approaches.

[0007] TIGIT (T cell immune receptor with Ig and ITIM domains), also known as Vstm3 and WUCAM, is a co-inhibitory receptor expressed on NK cells and CD8+ T cells, as well as a subset of CD4+ T cells, including immunosuppressive regulatory T cells (Tregs) [1-4]. Four known ligands for TIGIT exist: the poliovirus receptor (PVR), PVRL2, PVRL3, and PVRL4, all of which are overexpressed by tumor and antigen-presenting cells, leading to immunosuppression [3, 5-8]. These ligands also bind to the co-stimulatory molecule CD226 and the co-inhibitory molecules PVRIG and CD96 (CD96 is sometimes considered a co-stimulator). Antibodies against TIGIT disrupt the binding of TIGIT to its ligands and block its inhibitory signaling, shifting the balance in favor of CD226-mediated activation signaling, which induces a strong anti-tumor immune response [9-11]. In cancer and inflammatory diseases, TIGIT is upregulated and identified as a depletion marker [12-14], as are other depletion markers such as PD-1, LAG3, and TIM3 [15,16]. Furthermore, TIGIT has been identified as a key inhibitory receptor for a new population of T cells (stem cell-like memory T cells), potentially a preferred target for anti-PD-(L)1 efficacy [17,18]. TIGIT could be a promising therapeutic target for cancer immunotherapy, either as a single agent or in combination with other immunomodulators.

[0008] On the other hand, poliovirus receptor-related Ig domain containing (PVRIG, also known as CD112R) is one of the co-inhibitory immune checkpoint proteins. It plays an important role in reversing T cell exhaustion and increasing NK cell activation [19-22]. PVRIG belongs to the connexin and connexin-like family, whose members also include TIGIT, DNAM-1 (CD226), and CD96. PVRIG is expressed on NK cells and T cells, and is further upregulated on T cells after activation [19, 20]. The interaction between PVRIG and its ligand PVRL2 (CD112), expressed on APCs and various tumor cells, leads to the inhibition of T cell and NK cell activation [21, 22]. PVRL2 is also a ligand of CD226, which activates human T cells and NK cells after ligand interaction [23-25]. Despite the many similarities between PVRIG and TIGIT, scientists have reported significant differences that suggest these pathways are not redundant [20, 22]. Targeting these pathways can synergistically enhance anti-tumor responses.

[0009] Immune checkpoint blockade (anti-CTLA-4 antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies) may offer durable remissions for patients in a wide variety of cancers, such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), and triple-negative breast cancer (TNBC). However, despite this broad applicability, the majority (well over 80%) of cancer patients will eventually become refractory or resistant to CTLA-4, PD-1 / PD-L1 immune checkpoint therapies.

[0010] Therefore, there is a need for novel antibodies that can effectively target immune checkpoint molecules and restore T-cell function for cancer therapy. Summary of the Invention

[0011] This disclosure provides for these and other purposes. In a broader sense, this disclosure relates to providing compounds, methods, compositions, and articles of antibody with improved efficacy. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with other antibodies that respond to a variety of targets.

[0012] In some aspects, this disclosure provides peptide complexes, such as bispecific peptide complexes, that can bind to TIGIT and PVRIG proteins or antigens. Among other sources, TIGIT and PVRIG proteins can be of human, cynomolgus monkey, or mouse origin. In some embodiments, the peptide complex is cross-species reactive, for example, it can bind to human TIGIT, cynomolgus monkey TIGIT, and mouse TIGIT. In some embodiments, the peptide complex can bind to human PVRIG and cynomolgus monkey PVRIG.

[0013] In some embodiments, the peptide complex comprises a TIGIT-binding moiety and a PVRIG-binding moiety, wherein:

[0014] The TIGIT binding region comprises heavy chain CDRs (HCDR) 1, HCDR 2, and HCDR 3 and light chain CDRs (LCDR) 1, LCDR 2, and LCDR 3, wherein HCDR 1, HCDR 2, and HCDR 3 respectively contain the amino acid sequences of SEQ ID NO: 1, 2, and 3, and are substantially composed of or composed of the amino acid sequences of SEQ ID NO: 1, 2, and 3; and LCDR 1, LCDR 2, and LCDR 3 respectively contain the amino acid sequences of SEQ ID NO: 4, 5, and 6, and are substantially composed of or composed of the amino acid sequences of SEQ ID NO: 4, 5, and 6; and

[0015] The PVRIG binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences of SEQ ID NO: 7, 8, and 9, respectively, and are substantially composed of the amino acid sequences of SEQ ID NO: 7, 8, and 9 or are composed of the amino acid sequences of SEQ ID NO: 7, 8, and 9; and LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of SEQ ID NO: 10, 11, and 12, respectively, and are substantially composed of the amino acid sequences of SEQ ID NO: 10, 11, and 12 or are composed of the amino acid sequences of SEQ ID NO: 10, 11, and 12.

[0016] In some embodiments, the peptide complex comprises a TIGIT-binding moiety and a PVRIG-binding moiety, wherein:

[0017] The TIGIT assembly includes:

[0018] The heavy chain CDR (HCDR) 1 containing the amino acid sequence of SEQ ID NO: 1; the HCDR 2 containing the amino acid sequence of SEQ ID NO: 2; the HCDR 3 containing the amino acid sequence of SEQ ID NO: 3; the light chain CDR (LCDR) 1 containing the amino acid sequence of SEQ ID NO: 4; the LCDR 2 containing the amino acid sequence of SEQ ID NO: 5; and the LCDR 3 containing the amino acid sequence of SEQ ID NO: 6; and

[0019] The PVRIG integration component includes:

[0020] HCDR1 containing the amino acid sequence of SEQ ID NO: 7; HCDR2 containing the amino acid sequence of SEQ ID NO: 8; HCDR3 containing the amino acid sequence of SEQ ID NO: 9; light chain LCDR1 containing the amino acid sequence of SEQ ID NO: 10; LCDR2 containing the amino acid sequence of SEQ ID NO: 11; and LCDR3 containing the amino acid sequence of SEQ ID NO: 12.

[0021] In some implementations, the TIGIT integration portion includes:

[0022] (A) Heavy chain variable region (VH), which includes:

[0023] (i) The amino acid sequence shown in SEQ ID NO: 13;

[0024] (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 13; or

[0025] (iii) An amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) added, deleted, and / or substituted amino acids compared to SEQ ID NO: 13; and

[0026] (B) Light chain variable region (VL), which includes:

[0027] (i) The amino acid sequence as shown in SEQ ID NO: 14; or

[0028] (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 14; or

[0029] (iii) An amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) added, deleted and / or substituted amino acids compared to SEQ ID NO: 14.

[0030] In some implementations, the PVRIG binding portion includes:

[0031] (A) Heavy chain variable region (VH), which includes:

[0032] (i) The amino acid sequence as shown in SEQ ID NO: 15;

[0033] (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to that of SEQ ID NO: 15; or

[0034] (iii) An amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) added, deleted, and / or substituted amino acids compared to SEQ ID NO: 15; and

[0035] (B) Light chain variable region (VL), which includes:

[0036] (i) The amino acid sequence as shown in SEQ ID NO: 16; or

[0037] (ii) An amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 16; or

[0038] (iii) An amino acid sequence having one or more (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) added, deleted and / or substituted amino acids compared to SEQ ID NO: 16.

[0039] In some embodiments, the TIGIT binding portion includes HCDR1, HCDR2, and HCDR3 of the VH region as shown in SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 of the VL region as shown in SEQ ID NO: 14. In some embodiments, the PVRIG binding portion includes HCDR1, HCDR2, and HCDR3 of the VH region as shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 of the VL region as shown in SEQ ID NO: 16.

[0040] In some embodiments, the TIGIT binding portion comprises a VH region including the amino acid sequence of SEQ ID NO: 13 and a VL region including the amino acid sequence of SEQ ID NO: 14. In some embodiments, the PVRIG binding portion comprises a VH region including the amino acid sequence of SEQ ID NO: 15 and a VL region including the amino acid sequence of SEQ ID NO: 16.

[0041] In some embodiments, the TIGIT bonding portion is in the form of Fab or scFv. In some embodiments, the PVRIG bonding portion is in the form of Fab or scFv. In some embodiments, the TIGIT bonding portion is in the form of Fab, and the PVRIG bonding portion is in the form of scFv. In some embodiments, the TIGIT bonding portion is in the form of scFv, and the PVRIG bonding portion is in the form of Fab.

[0042] In some embodiments, the peptide complex further comprises a human IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 constant region. The human IgG constant region can be a native IgG Fc region or a variant thereof. In some embodiments, the peptide complex comprises a native human IgG1 Fc region.

[0043] In some implementations, variants of the Fc region contain one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or prolong antibody half-life.

[0044] In some embodiments, the peptide complex comprises one, two, or more TIGIT-binding moieties and one, two, or more PVRIG-binding moieties. The TIGIT-binding moieties may be the same or different, and / or the PVRIG-binding moieties may be the same or different. The peptide complex may be a homodimer or a heterodimer.

[0045] In some embodiments, the peptide complex comprises two heavy chains and two light chains, wherein the TIGIT-binding moiety is in the form of Fab and the PVRIG-binding moiety is in the form of scFv, and wherein from the N-terminus to the C-terminus:

[0046] The first and second heavy chains each contain structural domains operatively linked as in VH1-CH1-Fc-scFv or scFv-VH1-CH1-Fc.

[0047] The first and second light chains each contain structural domains that are operatively connected as in VL1-CL.

[0048] VH1-CH1 and VL1-CL originate from the TIGIT binding site, and scFv originates from the PVRIG binding site. VH1 and VL1 refer to the first VH and first VL in the polypeptide complex constituting the TIGIT binding site. VH2 and VL2 refer to the second VH and second VL constituting the PVRIG binding site.

[0049] In some embodiments, the peptide complex comprises two heavy chains and two light chains, wherein the TIGIT-binding moiety is in the form of scFv and the PVRIG-binding moiety is in the form of Fab, and wherein from the N-terminus to the C-terminus:

[0050] The first and second heavy chains each contain structural domains that are operatively linked as in scFv-VH2-CH1-Fc.

[0051] The first and second light chains each contain structural domains that are operatively connected as in VL2-CL.

[0052] scFv originates from the TIGIT binding region, while VH2-CH1 and VL2-CL originate from the PVRIG binding region.

[0053] In some embodiments, the peptide complex comprises two heavy chains and four light chains, wherein the TIGIT-binding and PVRIG-binding moieties are in the form of Fab, and wherein from the N-terminus to the C-terminus:

[0054] The first and second heavy chains each contain structural domains operatively linked in VH1-CH1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-CH1, VH1-CH1-VH2-CH1-Fc, or VH2-CH1-VH1-CH1-Fc.

[0055] The first and second light chains each contain structural domains that are operatively connected as in VL1-CL.

[0056] The third and fourth light chains each contain structural domains that are operatively connected as in VL2-CL.

[0057] VH1-CH1 and VL1-CL originate from the TIGIT binding region, while VH2-CH1 and VL2-CL originate from the PVRIG binding region.

[0058] In some embodiments, the polypeptide complex comprises two heavy chains and four light chains, wherein the TIGIT-binding and PVRIG-binding moieties are in the form of Fab, and the TIGIT-binding moieties are chimeric Fabs comprising VH1 operatively linked to a first T cell receptor (TCR) constant region (C1) and VL1 operatively linked to a second TCR constant region (C2), wherein from the N-terminus to the C-terminus:

[0059] The first and second heavy chains each contain structural domains operatively connected as in VH1-C1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-C1, VH1-C1-VH2-CH1-Fc, or VH2-CH1-VH1-C1-Fc.

[0060] The first and second light chains each contain structural domains that are operatively connected as in VL1-C2.

[0061] The third and fourth light chains each contain structural domains that are operatively connected as in VL2-CL.

[0062] VH1-C1 and VL1-C2 are derived from the TIGIT binding region, while VH2-CH1 and VL2-CL are derived from the PVRIG binding region. C1 and C2 refer to a pair of TCR constant regions that can bond together to form a dimer or their engineered variants containing one or more non-natural disulfide bonds.

[0063] In some embodiments, the polypeptide complex comprises two heavy chains and four light chains, wherein the TIGIT-binding and PVRIG-binding moieties are in the form of Fabs, and the PVRIG-binding moieties are chimeric Fabs comprising a VH2 operably linked to C1 and a VL2 operably linked to C2, wherein from the N-terminus to the C-terminus:

[0064] The first and second heavy chains each contain structural domains operatively connected in VH1-CH1-Fc-VH2-C1, VH2-C1-Fc-VH1-CH1, VH1-CH1-VH2-C1-Fc, or VH2-C1-VH1-CH1-Fc.

[0065] The first and second light chains each contain structural domains that are operatively connected as in VL1-CL.

[0066] The third and fourth light chains each contain structural domains that are operatively connected as in VL2-C2.

[0067] VH1-CH1 and VL1-CL are derived from the TIGIT binding region, while VH2-C1 and VL2-C2 are derived from the PVRIG binding region.

[0068] In some implementations, the scFv includes a VH region that is operatively connected to the VL region directly or via a connector, and the VH region is located at the N end of the VL region or the VL region is located at the N end of the VH region.

[0069] In some embodiments, one or more portions are operatively connected to the Fc region directly or via a connector. The connector can be a peptide connector, such as an intact or partial hinge region of an immunoglobulin (indicated herein as a “hinge”), or a conventionally used artificial connector, such as a GS connector. The GS connector, as used herein, can be (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, (GGSG)n, or (GGGSS)n, where n is an integer from 1 to 9. Preferably, the CH1 or C1 region, as described above, is operatively connected to the Fc region via a hinge region.

[0070] In some implementations, the polypeptide complex comprises:

[0071] (i) comprising the first heavy chain and the second heavy chain of SEQ ID NO: 17, and the first light chain and the second light chain of SEQ ID NO: 18; or

[0072] (ii) comprising the first heavy chain and the second heavy chain of SEQ ID NO: 19, and the first light chain and the second light chain of SEQ ID NO: 20.

[0073] In some respects, this disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of polypeptide complexes as disclosed herein.

[0074] In some respects, this disclosure provides expression vectors comprising nucleic acid molecules as disclosed herein.

[0075] In some respects, this disclosure provides host cells comprising expression vectors as disclosed herein.

[0076] In some respects, this disclosure provides pharmaceutical compositions comprising a polypeptide complex as disclosed herein and a pharmaceutically acceptable carrier.

[0077] In some aspects, this disclosure provides a method for preparing a polypeptide complex, comprising expressing the polypeptide complex in a host cell containing an expression vector encoding the polypeptide complex, and separating the polypeptide complex from a culture supernatant.

[0078] In some respects, this disclosure provides methods for modulating TIGIT / PVRIG-related immune responses in subjects, which include administering a peptide complex as disclosed herein to the subject.

[0079] In some aspects, this disclosure provides a method for inhibiting the growth of tumor cells in a subject, comprising administering to the subject, alone or in combination with another anticancer agent such as an antiPD-L1 antibody, an effective amount of a polypeptide complex or pharmaceutical composition as disclosed herein.

[0080] In some aspects, this disclosure provides methods for treating or preventing cancer or immune disorders in a subject, comprising administering to the subject an effective amount of a polypeptide complex as disclosed herein, either alone or in combination with another anticancer agent.

[0081] Anticancer agents can be chemotherapeutic agents, monoclonal antibodies, antibody-drug conjugates, etc. In some implementations, the anticancer agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0082] The cancers mentioned can be selected from colon cancer, lung cancer (such as NSCLC), breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, stomach cancer, pancreatic cancer, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, gastric cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, tumors of the central nervous system, mesothelioma, leukemia such as chronic lymphocytic leukemia, lymphoma such as diffuse large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma, myeloma, soft tissue cancer, and sarcoma. Immune disorders can be T-cell dysfunction disorders or infections.

[0083] In some implementations, such as the peptide complex disclosed herein, the peptide complex is administered in combination with an anti-PD-L1 antibody.

[0084] In some respects, this disclosure provides combinations of peptide complexes as disclosed herein with anti-PD-L1 antibodies such as atezolizumab.

[0085] In some aspects, this disclosure provides the use of polypeptide complexes (alone or in combination with another anticancer agent) as disclosed herein in the preparation of medicaments for the treatment or prevention of diseases such as cancer and immune disorders. In some embodiments, the anticancer agent is an antiPD-1 antibody or an antiPD-L1 antibody.

[0086] In some respects, this disclosure provides the use of peptide complexes as disclosed herein in the preparation of diagnostic agents for diagnosing diseases associated with TIGIT / PVRIG overexpression.

[0087] In some aspects, this disclosure provides polypeptide complexes as disclosed herein for the treatment or prevention of cancer and immune disorders. In some embodiments, antibodies as disclosed herein are used in combination with PD-1 / PD-L1 antagonists such as PD-L1 antibodies.

[0088] In some respects, this disclosure provides kits or devices that contain a polypeptide complex as disclosed herein in one or more containers.

[0089] The foregoing is a summary of the invention and therefore necessarily contains simplifications, generalizations, and omissions of details; thus, those skilled in the art will understand that the summary is merely illustrative and not intended to be limiting in any way. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to serve as an aid in determining the scope of the claimed subject matter. Attached Figure Description

[0090] Figure 1 A schematic diagram showing the structures of W3XX104-T4U1.G15-2.uIgG1 and W3XX104-T4U1.G17-2.uIgG1 is shown.

[0091] Figure 2-7 The antibody and human TIGIT were shown to be compatible, as determined by FACS. Figure 2 ) and human PVRIG ( Figure 3 ), crab-eating macaques (TIGIT) Figure 4 ), cynomolgus monkey PVRIG ( Figure 5 ), mouse TIGIT ( Figure 6 ) and mouse PVRIG ( Figure 7 The combination of ).

[0092] Figure 8 The SPR sensing map of the antibody that binds to human TIGIT is shown.

[0093] Figure 9 The SPR sensing map of the antibody that binds to human PVRIG is shown.

[0094] Figure 10 The binding of antibodies to TIGIT and PVRIG paralogs, as determined by ELISA, was shown.

[0095] Figure 11 The results show the dual binding of the antibody to human TIGIT and PVRIG.

[0096] Figure 12 The results show the results of antibody blocking the binding of PVR to TIGIT, as determined by FACS.

[0097] Figure 13 The results show the effects of antibody blocking the binding of PVRL2 to PVRIG, as determined by ELISA.

[0098] Figure 14 The results of the antibody in the Jurkat TIGIT / PVRIG / NFAT-luciferase reporter assay are shown.

[0099] Figure 15-16 The antibody was shown to have an effect on NK cell killing assay ( Figure 15) and T cell activation assay ( Figure 16 The effect in ).

[0100] Figures 17A-17B It showed W3XX104-T4U1.G17-2.uIgG1 ( Figure 17A ) and W3XX104-T4U1.G15-2.uIgG1 ( Figure 17B The melting curve of ).

[0101] Figures 18A-18B It showed W3XX104-T4U1.G17-2.uIgG1 ( Figure 18A ) and W3XX104-T4U1.G15-2.uIgG1 ( Figure 18B A graph of the diffusion coefficient for concentration.

[0102] Figures 19A-19B The HIC-HPLC showed W3XX104-T4U1.G17-2.uIgG1 ( Figure 19A ) and W3XX104-T4U1.G15-2.uIgG1 ( Figure 19B ) is retained.

[0103] Figure 20 The study showed changes in monkey body weight during pharmacokinetic studies.

[0104] Figure 21-24 This shows the presence of W3XX104-T4U1.G17-2.uIgG1 in the pharmacokinetic studies in monkeys. Figure 21 and Figure 22 ) and W3XX104-T4U1.G15-2.uIgG1 ( Figure 23 and Figure 24 Overview of the competition.

[0105] Figure 25 The average change in tumor volume in the CT26 mouse model is shown.

[0106] Figure 26 The changes in individual tumor volume after treatment in different groups are shown.

[0107] Figure 27 The tumor weights of different groups at the study endpoint (D17) are shown in the CT26 mouse model.

[0108] Figure 28 The mean weight changes in different groups after administration in the CT26 mouse model are shown. Detailed Implementation

[0109] While the invention may be embodied in many different forms, this document discloses specific illustrative embodiments that illustrate the principles of the invention. It should be emphasized that the invention is not limited to the specific illustrative embodiments. Furthermore, any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

[0110] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention shall have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. More specifically, as used in this specification and the appended claims, unless the context explicitly requires otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “comprises” and “comprised” is not restrictive. Moreover, the scope provided in the specification and appended claims includes both endpoints and all points in between.

[0111] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques disclosed herein are generally performed according to conventional methods well-known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbaset al., Cellular and Molecular Immunology, 6 thed., WBSaunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The nomenclature, laboratory procedures, and techniques used in combination with those described herein in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry are those well-known and commonly used in the art.

[0112] definition

[0113] To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0114] As used herein, the term “antibody” or “Ab” is used in the broadest sense and covers any form of antibody that exhibits the desired biological or binding activity. It covers, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single-domain antibodies. Bispecific polypeptide complexes as disclosed herein also belong to the antibody family. Common antibodies typically consist of heavy and light chains. Heavy chains can be classified as μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). As shown herein, the constant regions can be modified in various ways, or they can be replaced with constant regions derived from other immunoglobulins. The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions (CDRs)), which are separated by relatively conserved regions (called frame regions (FRs)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The extent of the frame regions and CDRs can be precisely identified using methods known in the art, such as the Kabat definition, Chothia definition, AbM definition, EU definition, and / or Contact definition, all of which are well known in the art. See, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; Edelman et al., Proc Natl Acad Sci US A. 1969 May, 63(1):78-85; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs.Correspondences or comparisons between different defined numberings can be found, for example, at www.imgt.org / (see also Giudicelli V et al. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. (1997) 25:206–11; and Lefranc MP et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol. (2003) 27:55–77). Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0115] As used herein, the term "antigen-binding moiety" refers to an antibody fragment formed from a portion of an antibody containing one or more CDRs, or any other antibody fragment that binds to an antigen but does not contain the complete structure of the native antibody. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, biantibodies, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv fragments (scFv), disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), multispecific antibodies, camelified single-domain antibodies, single variable domain (i.e., VHH), nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding moiety is capable of binding to the same antigen that the parent antibody binds to. In some embodiments, the antigen-binding moiety may contain one or more CDRs from a particular antibody that have been grafted into a framework region from a different antibody. A more detailed description of the antigen-binding portion is found in Spiess et al, (2015) Molecular Immunology 67: 95-106, and Brinkman et al., mAbs, 9(2), pp.182–212 (2017), which are incorporated herein by reference in their entirety.

[0116] The term "Fab" in antibody refers to a portion of the antibody consisting of a single light chain (both the variable and constant regions) associated with a single heavy chain via disulfide bonds. In some embodiments, the constant regions of both the light and heavy chains of the Fab may be replaced with TCR constant regions Cα and Cβ or engineered variants thereof.

[0117] The “Fc” (short for crystallizable fragment) in antibodies refers to the portion of the antibody containing the second (CH2) and third (CH3) constant regions of the first heavy chain associated with the second and third constant regions of the second heavy chain via disulfide bonds. The Fc is a specific dimerizing domain. As used herein, the Fc region may also contain part or all of the hinge region. The Fc region of an antibody is responsible for various effector functions, such as ADCC and CDC, but typically does not function in antigen binding. The ability of an antibody to initiate and regulate effector functions through its Fc domain is a key component of its protective activity in vivo.

[0118] As used herein, the term "monoclonal antibody" or "mAb" refers to a formulation of an antibody molecule consisting of a single molecular unit. Monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope.

[0119] As used herein, the term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species (such as rats or mice) has been grafted onto a human frame sequence. Further frame region modifications may be made within the human frame sequence. Humanized antibodies may optionally also contain at least a portion of an immunoglobulin constant region (e.g., Fc), typically the constant region of human immunoglobulins.

[0120] As used herein, the term "human antibody" or "fully human antibody" is intended to include antibodies with variable regions, where both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence.

[0121] Unless otherwise stated, the term "TIGIT" or "T-cell immune receptor with Ig and ITIM domains" as used herein refers to any naturally occurring TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). TIGIT is also known in the art as DKFZp667A205, FLJ39873, protein 9 containing V-set and immunoglobulin domains, protein 3 containing V-set and transmembrane domains, VSIG9, VSTM3, and WUCAM. The term encompasses full-length unprocessed TIGIT, the extracellular domains of TIGIT, and any form of TIGIT produced by cellular processing. The term also encompasses variants of naturally occurring TIGIT, such as splice variants or allelic variants.

[0122] The term “PVRIG” or “protein containing the poliovirus receptor-associated immunoglobulin domain” includes known or wild-type PVRIGs, or variants, conjugates, or fragments thereof (particularly extracellular domain fragments). PVRIGs are transmembrane domain proteins possessing a signal peptide, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. PVRIGs are expressed on the cell surface of NK cells and T cells and share several similarities with other known immune checkpoints. Identification and methods used to demonstrate that PVRIGs are checkpoint receptors can be found in WO2016 / 134333, which is incorporated herein by reference.

[0123] As used herein, the term "PD-1 / PD-L1 antagonist" includes PD-L1 antagonists (such as anti-PD-L1 antibodies) that reduce, block, inhibit, eliminate, or interfere with signal transduction caused by one or more interactions between PD-L1 and its binding partners (such as PD-1 or B7-1), as well as PD-1 antagonists (such as anti-PD-1 antibodies) that reduce, block, inhibit, eliminate, or interfere with signal transduction caused by one or more interactions between PD-1 and its binding partners (such as PD-L1, PD-L2). In some implementations, the PD-1 antagonist is an anti-PD-1 antagonist antibody selected from, but not limited to, the following: nivolumab (MDX-1106) or pembrolizumab (formerly known as lambolizumab (MK-3475), MED1-0680, PDR001 (spartalizumab), REGN2810 (cimiplimab), BGB-108, prolgolimab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostarlimab, retifanlimab, spartalizumab. umab), sasanlimab, penpulimab, CS1003, HLX10, SCT-I10A, SHR-1316, CS1001, envafolimab, TQB2450, ZKAB001, LP-002, zimberelimab, balstilimab, genolimzumab, BI 754091, cetrelimab, YBL-006, BAT1306, HX008, CX-072, IMC-001, KL-A167, budigalimab, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, APL-502, cosibelimab, lodapolimab, GS-4224, INCB086550, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, MAX-10181, RC98, BION-004, AM0001, CB201, ENUM 244C8, ENUM 388D4, AUNP-012, STI-1110, ADG104, AK-103, LBL-006, hAb21, AVA-004, PDL-GEX, INCB090244, KD036, KY1003, LYN192, MT-6035, VXM10, YBL-007, ABSK041, GB7003, JS-003, and HS-636. In some embodiments, the PD-L1 antagonist is an anti-PD-L1 antagonist antibody selected from, but not limited to, the following: MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab). PD-1 / PD-L1 antagonists include known antibodies and internally developed antibodies.

[0124] The terms "operably link" and "operably linked" refer to the juxtaposition of two or more biological sequences of interest in such a manner, with or without spacers or linkers, that they are in a relationship that allows them to function in the intended way. When used with respect to peptides, it is intended to mean that the peptide sequences are linked in a manner that allows the linked product to have the intended biological function. For example, the variable region of an antibody can be operably linked to a constant region to provide a stable product with antigen-binding activity. When one chain of an antigen-binding moiety comprising two amino acid chains (such as antibody Fab) is linked to one chain of an Fc region in such a manner that both can perform their respective functions, the moiety can be considered "operably linked" to the Fc region. The term can also be used with respect to polynucleotides. For example, when a polynucleotide encoding a peptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), it is intended to mean that the polynucleotide sequence is linked in a manner that allows the peptide to be expressed from the regulated polynucleotide. When used in this document to describe the domains of a peptide, the term "operable link" may be indicated by "-" and may refer to a direct link between domains or a link via a linker comprising 1-30 amino acids of length, such as a single amino acid or a series of (G4S)n linkers, where n = 1-5 (1, 2, 3, 4 or 5).

[0125] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or its antigen-binding moiety and an antigen). Binding affinity between two molecules can be quantified by determining the equilibrium dissociation constant (KD). Accordingly, KD can be determined by measuring the kinetics of complex formation and dissociation using a surface plasmon resonance (SPR) method (Biacore™) as a non-limiting example. The rate constants corresponding to association and dissociation of a monovalent complex are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. The term ka (or kon) refers to the association rate of a specific antibody-antigen interaction, while the term kd (or koff) refers to the dissociation rate of a specific antibody-antigen interaction. KD is related to ka and kd via the equation KD = Kd / ka or koff / kon. Antibody binding kinetics and binding affinity can be evaluated using standard assays known in the art or as described in the Examples section below.

[0126] As used in this article, the term "EC" 50 The term "IC50," also known as "half-maximal effective concentration," refers to the concentration of a drug, antibody, or toxin that, after a specified exposure time, induces an intermediate response between baseline and maximum. As used herein, the term "IC50" is... 50The term "half-maximal inhibitory concentration" (MCC), also known as the "half-maximal inhibitory concentration," refers to the half-maximal inhibitory concentration of a drug, antibody, or other substance. It is a measure of the effectiveness of a drug, antibody, or other substance in inhibiting biological or biochemical functions. In the context of this application, EC... 50 and IC 50 It is expressed in units of “nM” or “M”.

[0127] As used herein, the ability to "inhibit binding" or "block binding" refers to the ability of an antibody to inhibit the binding interaction between two molecules (e.g., human TIGIT and PVR, human PVRIG and PVRL2) to any detectable level. In some embodiments, the bispecific peptide complexes disclosed herein are used at IC50 values ​​not exceeding 1 nM, 0.8 nM, 0.6 nM, 0.4 nM, or 0.3 nM. 50 Blocking the binding between human TIGIT and PVR. In some embodiments, the bispecific peptide complex, as disclosed herein, is used at an IC50 concentration not exceeding 1 nM, 0.9 nM, or 0.8 nM. 50 Blocks the binding between human PVRIG and PVRL2.

[0128] As used herein, the term "separated" refers to a state obtained artificially from the natural state. If a "separated" substance or component exists in nature, it may be because its natural environment has changed, or the substance has been separated from its natural environment, or both. For example, an unseparated polynucleotide or polypeptide may naturally exist in a living animal, and a highly pure copy of the same polynucleotide or polypeptide separated from this natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude the presence of artificially or synthetically produced mixtures, nor does it exclude other impurities that do not affect the activity of the separated substance.

[0129] As used herein, the term "vector" refers to a nucleic acid medium that may have intercalated polynucleotides. When a vector allows the expression of a protein encoded by an intercalated polynucleotide, it is called an expression vector. A vector may carry genetic material elements that are expressed in a host cell through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0130] As used herein, the term "host cell" refers to a cellular system that can be engineered to produce proteins, protein fragments, or peptides of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term covers not only the specific subject cell but also its progeny. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may differ from the parent cell, but they are still included within the scope of the term "host cell."

[0131] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of sequences. "Identity percentage" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules, and it is calculated based on the size of the smallest of the compared molecules. For these calculations, gaps in the alignment (if any) are preferably resolved using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al, 1988, SIAMJ. Applied Math. 48:1073.

[0132] As used herein, the term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells, ultimately leading to the production of immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the organism's immune system after stimulation with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in a host depends on three factors: the characteristics of the antigen, the host's reactivity, and the immunization method.

[0133] As used herein, the term "transfection" refers to the process by which nucleic acids are introduced into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In a particular embodiment of the invention, the human TIGIT gene is transfected into 293F cells.

[0134] As used herein, the term “SPR” or “surface plasmon resonance” refers to, and includes, optical phenomena that allow for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Example 5 and Jönsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0135] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers (one cell at a time) based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence-Activated Cell Sorting". Retrieved 2017-11-09.). Instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, Calif.), the Epics C from Coulter EpicsDivision (Hialeah, Fla.), and the MoFlo from Cytomation (Colorado Springs, Colo.).

[0136] The terms “subject” and “patient” are used interchangeably and include mammals, such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammal species. The term does not necessarily indicate that a subject has been diagnosed with a specific disease, but generally refers to an individual under medical supervision.

[0137] As used herein, the terms “prevent,” “preventing,” or “prevention” in the context of preventing a condition generally refer to preventing or delaying the onset of a disease in a subject (whether human or animal), or preventing the clinical or subclinical manifestation of a disease in a subject (whether human or animal), for example, preventing the disease from occurring in subjects who are susceptible to the condition or disease but have not yet been diagnosed with it.

[0138] As used herein, the terms “treatment,” “treating,” or “treated” in the context of treating a condition generally refer to treatment and therapies (whether for humans or animals) that achieve some desired therapeutic effect, such as inhibiting the progression of the disease, and including a reduction in the rate of progression, cessation of the rate of progression, regression of the condition, improvement of the condition, and cure of the condition. In the context of cancer, “treatment” can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof.

[0139] As used herein, the term "effective amount" refers to an amount of an active compound or a material, composition, or dosage form containing an active compound that, when administered according to the desired treatment regimen, is effective in producing some desired therapeutic effect commensurate with a reasonable benefit / risk ratio. For example, when used in connection with the treatment of a disease or condition, "effective amount" refers to an amount or concentration of an antibody or its antigen-binding portion that is effective in treating said disease or condition.

[0140] As used herein, the term "pharmaceutically acceptable" means that the medium, diluent, excipient and / or its salt are chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0141] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0142] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with an antigen or when delivered to an organism beforehand, can enhance the immune response to the antigen or alter the type of immune response in the organism. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium parvum, lipopolysaccharides, cytokines, etc. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0143] Peptide complex targeting TIGIT and PVRIG

[0144] The polypeptide complexes described herein include bispecific antibodies and their antigen-binding moieties. As used herein, the term "polypeptide complex" is used interchangeably with "antibody." In some embodiments, the bispecific antibody and its antigen-binding moieties have a first specificity against TIGIT (e.g., human, cynomolgus monkey, and mouse TIGIT) and a second specificity against PVRIG (e.g., human and cynomolgus monkey PVRIG). Such antibodies may be referred to herein as, for example, "anti-TIGIT / anti-PVRIG," "anti-PVRIG / TIGIT," "anti-PVRIGxTIGIT," or "PVRIGxTIGIT" bispecific antibodies, or other similar terms.

[0145] In some embodiments, the bispecific antibody described herein comprises a first antigen-binding moiety (PVRIG-binding moiety) that specifically binds to PVRIG and a second antigen-binding moiety (TIGIT-binding moiety) that specifically binds to TIGIT. To construct the bispecific antibody, considering the stability, expression level, binding capacity, and other functions of the assembled antibody, the first and second antigen-binding moieties can be in the form of Fab, scFv, or VHH. For example, the TIGIT-binding moiety can be in the form of Fab, and the PVRIG-binding moiety can be in the form of scFv; or, the PVRIG-binding moiety can be in the form of Fab, and the TIGIT-binding moiety can be in the form of scFv. In some embodiments, both the TIGIT-binding moiety and the PVRIG-binding moiety are in the form of Fab.

[0146] In some embodiments, bispecific antibodies, such as those disclosed herein, comprise more than one antigen-binding moiety that specifically binds to PVRIG and / or more than one antigen-binding moiety that specifically binds to TIGIT. Typically, for bispecific antibodies, the more than one antigen-binding moiety has the same variable region (and thus targets the same antigen / epitope), or is identical in both the variable and constant regions (if present). For example, an antibody may comprise two identical PVRIG-binding moieties and one TIGIT-binding moieties, or one PVRIG-binding moieties and two identical TIGIT-binding moieties, or two identical PVRIG-binding moieties and two identical TIGIT-binding moieties. Furthermore, when two PVRIG-binding moieties or two TIGIT-binding moieties are present, these moieties may take the form of: (i) the TIGIT-binding moieties are Fab and the PVRIG-binding moieties are scFv, (ii) the TIGIT-binding moieties are scFv and the PVRIG-binding moieties are Fab, or (iii) both the TIGIT-binding moieties and the PVRIG-binding moieties are Fab.

[0147] In some specific embodiments, the TIGIT binding portion is in the form of Fab, comprising a first heavy chain variable domain (VH1) operatively linked to the CH1 domain of the antibody heavy chain (VH1-CH1) and a first light chain variable domain (VL1) operatively linked to the constant (CL) domain of the antibody light chain (VL1-CL), and the PVRIG binding portion is in the form of scFv, comprising a second heavy chain variable domain (VH2) operatively linked to a second light chain variable domain (VL2) (VH2-VL2). The PVRIG-binding scFv can be located at the N-terminus or C-terminus of the heavy or light chain of the bispecific polypeptide complex, and within the scFv, VH2 can be located at the N-terminus of VL2, or vice versa.

[0148] In some specific embodiments, the TIGIT-binding portion is in the form of an scFv containing a first VH (VH1-VL1) operatively linked to a first VL, and the PVRIG-binding portion is in the form of a Fab containing a second VH (VH2-CH1) operatively linked to a CH1 domain of the antibody heavy chain and a second VL (VL2-CL) operatively linked to a constant (CL) domain of the antibody light chain. The TIGIT-binding scFv can be located at the N-terminus or C-terminus of the heavy or light chain of the bispecific polypeptide complex, and within the scFv, VH1 can be located at the N-terminus of VL1, or vice versa.

[0149] As an example, the bispecific peptide complex disclosed herein may comprise two heavy chains and two light chains, wherein the TIGIT-binding moiety is in the form of Fab and the PVRIG-binding moiety is in the form of scFv, and wherein from the N-terminus to the C-terminus:

[0150] (a) The first heavy chain and the second heavy chain each contain structural domains operatively connected as in VH1-CH1-hinge-Fc-scFv, and the first light chain and the second light chain each contain structural domains operatively connected as in VL1-CL; or

[0151] (b) The first heavy chain and the second heavy chain each contain structural domains operatively connected as in scFv-VH1-CH1-hinge-Fc, and the first light chain and the second light chain each contain structural domains operatively connected as in VL1-CL.

[0152] VH1-CH1 and VL1-CL are derived from the TIGIT binding region, and scFv (VH2-VL2 or VL2-VH2 from the N end to the C end) is derived from the PVRIG binding region.

[0153] In another example, a bispecific polypeptide complex as disclosed herein may comprise two heavy chains and two light chains, wherein the TIGIT-binding moiety is in the form of scFv and the PVRIG-binding moiety is in the form of Fab, and wherein from the N-terminus to the C-terminus:

[0154] (a) The first heavy chain and the second heavy chain each contain structural domains operatively connected as in VH2-CH1-hinge-Fc-scFv, and the first light chain and the second light chain each contain structural domains operatively connected as in VL2-CL; or

[0155] (b) The first heavy chain and the second heavy chain each contain structural domains operatively connected as in scFv-VH2-CH1-hinge-Fc, and the first light chain and the second light chain each contain structural domains operatively connected as in VL2-CL.

[0156] Among them, scFv (VH1-VL1 or VL1-VH1 from N end to C end) comes from the TIGIT binding part, and VH2-CH1 and VL2-CL come from the PVRIG binding part.

[0157] In some specific embodiments, the TIGIT binding portion is in the form of a Fab, comprising a first VH (VH1-CH1) operatively linked to the CH1 domain of the antibody heavy chain, and a first VL (VL1-CL) operatively linked to the constant domain of the antibody light chain (CL). The PVRIG binding portion is also in the form of a Fab, comprising a second heavy chain variable domain (VH2-CH1) operatively linked to the CH1 domain of the antibody heavy chain, and a second VL (VL2-CL) operatively linked to the constant domain of the antibody light chain (CL). The TIGIT binding portion may be located at the N-terminus of the Fc region and the PVRIG binding portion may be located at the C-terminus of the Fc region, or the TIGIT binding portion may be located at the C-terminus of the Fc region and the PVRIG binding portion may be located at the N-terminus of the Fc region, i.e., the Fc region is located between the TIGIT binding portion and the PVRIG binding portion. In some other embodiments, both the TIGIT binding portion and the PVRIG binding portion are located at the N-terminus of the Fc region. For dimerization stability, the CH1 and CL domains of the TIGIT-binding or PVRIG-binding regions can be replaced by a pair of TCR constant regions to differentiate between the TIGIT-binding and PVRIG-binding Fabs. Therefore, in some embodiments, the CH1 domain is replaced by a first TCR constant region (C1, i.e., the wild-type or engineered TCRβ chain constant region), and the CL domain is replaced by a second TCR constant region (C2, i.e., the wild-type or engineered TCRα chain constant region). Alternatively, they can be interchanged, with the TCRβ chain constant region in the light chain and the TCRα chain constant region in the heavy chain.

[0158] TCR (T cell receptor) is a heterodimeric T cell surface protein belonging to the immunoglobulin superfamily and is similar to a hapten with a single heavy chain and a single light chain. The native TCR has extracellular, transmembrane, and intracellular portions. The extracellular domain of the TCR has a proximal membrane constant region and a distal membrane variable region. Introducing the TCR constant region to replace the commonly used CH1 and CL domains has been shown to increase the stability and expression levels of the resulting antibody form. A detailed description of the utility of the TCR constant region in assembling two parental antibodies into bispecific molecules with desired valence and function is disclosed in WO2019 / 057122, the entire contents of which are incorporated herein by reference. The replacement of the TCR constant region yields chimeric Fabs with a unique light-heavy chain interface orthogonal to the interface of conventional antibody Fabs. The assembly of different forms of chimeric Fabs and conventional Fabs can produce a variety of bispecific molecules with different structures and valences.

[0159] The chimeric Fab comprises a pair of TCR constant regions, specifically TCRα and TCRβ constant regions (wild-type or preferably engineered), in the light and heavy chains, respectively. In some embodiments, the chimeric Fab includes a first TCR constant region and a second TCR constant region bonded via non-natural interchain disulfide bonds. The TCR constant regions may be engineered to introduce non-natural disulfide bonds between the light and heavy chain interfaces. In some other embodiments, the chimeric Fab includes a pair of wild-type TCR constant regions, such as wild-type human TCRβ and TCRα constant regions.

[0160] The sequences of the constant regions of the wild-type human TCRβ and TCRα chains can be found in NCBI accession number A0A5B9 ( www.uniprot.org / uniprot / A0A5B9 ) and NCBI Registry Number P01848 ( www.uniprot.org / uniprot / P01848The pair of TCR constant regions used to construct the bispecific antibody described herein are derived from the wild-type TCR constant region and have one or more substitutions, additions, or deletions of one or more amino acids. The bispecific antibody may comprise an engineered TCRβ chain constant region and an engineered TCRα chain constant region (referred to herein as C1 and C2). As shown in this application, a bispecific antibody may comprise an engineered TCR β chain constant region (C1) having the sequence shown in SEQ ID NO: 21 (LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR) and an engineered TCR α chain constant region (C2) having the sequence shown in SEQ ID NO: 22 (PDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS). Various TCR constant region variants for constructing bispecific antibody forms have been disclosed in PCT / CN2021 / 072601, the entire contents of which are incorporated herein by reference.

[0161] As an example, the bispecific peptide complex disclosed herein comprises two heavy chains and four light chains, wherein the TIGIT-binding moiety is in the form of Fab, and the PVRIG-binding moiety is in the form of Fab, wherein from the N-terminus to the C-terminus:

[0162] The first and second heavy chains each contain structural domains operatively connected as in VH1-C1-VH2-CH1-hinge-Fc or VH2-CH1-VH1-C1-hinge-Fc (VH1-C1 from the TIGIT joint and VH2-CH1 from the PVRIG joint); the two light chains contain structural domains operatively connected as in VL1-C2 (VL1-C2 from the TIGIT joint); and the other two light chains contain structural domains operatively connected as in VL2-CL (VL2-CL from the PVRIG joint).

[0163] As another example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, wherein the TIGIT-binding moiety is in the form of Fab, and the PVRIG-binding moiety is in the form of Fab, wherein from the N-terminus to the C-terminus:

[0164] The first and second heavy chains each contain structural domains operatively connected as in VH1-C1-hinge-Fc-VH2-CH1 or VH2-CH1-hinge-Fc-VH1-C1 (VH1-C1 from the TIGIT joint and VH2-CH1 from the PVRIG joint); the two light chains contain structural domains operatively connected as in VL1-C2 (VL1-C2 from the TIGIT joint); and the other two light chains contain structural domains operatively connected as in VL2-CL (VL2-CL from the PVRIG joint).

[0165] As a further example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, wherein the TIGIT-binding moiety is in the form of Fab, and the PVRIG-binding moiety is in the form of Fab, wherein from the N-terminus to the C-terminus:

[0166] The first and second heavy chains each contain structural domains operatively connected as in VH1-CH1-VH2-C1-hinge-Fc or VH2-C1-VH1-CH1-hinge-Fc (VH1-CH1 from the TIGIT junction and VH2-C1 from the PVRIG junction); the two light chains contain structural domains operatively connected as in VL1-CL (VL1-CL from the TIGIT junction); and the other two light chains contain structural domains operatively connected as in VL2-C2 (VL2-C2 from the PVRIG junction).

[0167] As a further example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, wherein the TIGIT-binding moiety is in the form of Fab, and the PVRIG-binding moiety is in the form of Fab, wherein from the N-terminus to the C-terminus:

[0168] The first and second heavy chains each contain structural domains operatively connected as in VH1-CH1-hinge-Fc-VH2-C1 or VH2-C1-hinge-Fc-VH1-CH1 (VH1-C1 from the TIGIT joint and VH2-C1 from the PVRIG joint); the two light chains contain structural domains operatively connected as in VL1-CL (VL1-CL from the TIGIT joint); and the other two light chains contain structural domains operatively connected as in VL2-C2 (VL2-C2 from the PVRIG joint).

[0169] In some implementations, such as the bispecific antibodies disclosed herein, each antibody comprises one TIGIT-binding moiety and two PVRIG-binding moiety, or each antibody comprises two TIGIT-binding moiety and one PVRIG-binding moiety. These moiety may be in the form of Fab or scFv.

[0170] As an example, the bispecific peptide complex described in this paper may contain two heavy chains and two light chains, wherein the TIGIT-binding moiety is in the form of Fab and the PVRIG-binding moiety is in the form of scFv, and wherein from the N-terminus to the C-terminus:

[0171] The first heavy chain contains structural domains operatively connected as in VH1-CH1-hinge-Fc-scFv or scFv-VH1-CH1-hinge-Fc; the second heavy chain contains structural domains operatively connected as in VH1-CH1-hinge-Fc; the two light chains contain structural domains operatively connected as in VL1-CL.

[0172] VH1-CH1 and VL1-CL are derived from the TIGIT binding region, and scFv (VH2-VL2 or VL2-VH2 from the N end to the C end) is derived from the PVRIG binding region.

[0173] As another example, the bispecific peptide complex described in this paper may contain two heavy chains and two light chains, wherein the TIGIT binding moiety is in the form of scFv and the PVRIG binding moiety is in the form of Fab, and wherein from the N-terminus to the C-terminus:

[0174] The first heavy chain contains structural domains operatively connected as in VH2-CH1-hinge-Fc-scFv or scFv-VH2-CH1-hinge-Fc; the second heavy chain contains structural domains operatively connected as in VH2-CH1-hinge-Fc; the two light chains contain structural domains operatively connected as in VL2-CL.

[0175] VH2-CH1 and VL2-CL are derived from the PVRIG binding region, and scFv (VH1-VL1 or VL1-VH1 from the N end to the C end) is derived from the TIGIT binding region.

[0176] Depending on the bispecific form and / or numbering convenience, such as the numbering sequences disclosed herein, such as first or second, they may be different. For example, a first VH may be numbered as a second VH, and a first VL may be numbered as a second VL. As another example, depending on preference and / or convenience, a second antigen-binding portion may be numbered as a first antigen-binding portion, and a first antigen-binding portion may be numbered as a second antigen-binding portion.

[0177] CDR and variable region of bispecific peptide complex

[0178] In some embodiments, the bispecific polypeptide complex or its antigen-binding moiety comprises a first antigen-binding moiety specifically binding to TIGIT (preferably human TIGIT) and a second antigen-binding moiety specifically binding to PVRIG (preferably human PVRIG), wherein the first and second antigen-binding moieties are derived from an anti-TIGIT antibody and an anti-PVRIG antibody, respectively. The parent antibody may be developed and publicly known, or it may be developed de novo. The term "derived from" generally means that the antigen-binding moiety comprises the CDR sequence of the parent antibody or a highly homologous CDR sequence thereof, and preferably, a variable region of the parent antibody. The antigen-binding moiety may also comprise a variant of the parent antibody CDR sequence that retains antigen-binding specificity. For example, one or two amino acids in one or more CDR regions may be modified to reduce the risk of glycosylation and deamidation compared to the original CDR sequence of the parent antibody.

[0179] Specifically, in bispecific antibodies as illustrated herein, the TIGIT binding moiety includes:

[0180] A) Select one or more heavy chain CDRs (HCDRs) from the following groups:

[0181] (i) HCDR1, which contains the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having an addition, deletion and / or substitution of no more than 1, 2 or 3 amino acids compared to SEQ ID NO: 1;

[0182] (ii) HCDR2, comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having an addition, deletion, and / or substitution of no more than 1, 2, or 3 amino acids compared to SEQ ID NO: 2; and

[0183] (iii) HCDR3, which contains the amino acid sequence of SEQ ID NO: 3 or has an amino acid sequence with no more than 1, 2 or 3 added, deleted and / or substituted amino acids compared to SEQ ID NO: 3;

[0184] B) Select one or more light chain CDRs (LCDRs) from the following groups:

[0185] (i) LCDR1, which contains the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having an addition, deletion and / or substitution of no more than 1, 2 or 3 amino acids compared to SEQ ID NO: 4;

[0186] (ii) LCDR2, comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having an addition, deletion, and / or substitution of no more than 1, 2, or 3 amino acids compared to SEQ ID NO: 5; and

[0187] (iii) LCDR3, comprising the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having an addition, deletion, and / or substitution of no more than 1, 2, or 3 amino acids compared to SEQ ID NO: 6; or

[0188] C) One or more HCDRs of A) and one or more LCDRs of B); and

[0189] The PVRIG integration component includes:

[0190] A') Select one or more HCDRs from the following groups:

[0191] (i) HCDR1, which contains the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having an addition, deletion and / or substitution of no more than 1, 2 or 3 amino acids compared to SEQ ID NO: 7;

[0192] (ii) HCDR2, comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having an addition, deletion, and / or substitution of no more than 1, 2, or 3 amino acids compared to SEQ ID NO: 8; and

[0193] (iii) HCDR3, which contains the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having an addition, deletion and / or substitution of no more than 1, 2 or 3 amino acids compared to SEQ ID NO: 9;

[0194] B') Select one or more LCDRs from the following groups:

[0195] (i) LCDR1, which contains the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having an addition, deletion and / or substitution of no more than 1, 2 or 3 amino acids compared to SEQ ID NO: 10;

[0196] (ii) LCDR2, comprising the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having an addition, deletion, and / or substitution of no more than 1, 2, or 3 amino acids compared to SEQ ID NO: 11; and

[0197] (iii) LCDR3, comprising the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having an addition, deletion, and / or substitution of no more than 1, 2, or 3 amino acids compared to SEQ ID NO: 12; or

[0198] One or more HCDRs of C') and A') and one or more LCDRs of B').

[0199] In some implementations, the CDR of the anti-TIGIT portion is determined according to the Contact definition, while the CDR of the anti-PVRIG portion is determined according to the IMGT / Kabat definition. Variable regions and CDRs in the antibody sequence can be determined according to general rules developed in the art or by comparing the sequence against a database of known variable regions. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDR of antibodies as disclosed herein is intended to be within the scope of this application. Although the CDRs shown in Table A below are determined by the Contact or Kabat and IMGT numbering system, CDRs can also be determined using Chothia, MacCallum, and other methods known in the art. Methods for determining these regions are described, for example, in Kontermann and Dubel, eds., Antibody Engineering, Springer, New York, NY, 2001 and Dinarello et al., Current Protocols in Immunology, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described below and can be accessed through the following: the “Abysis” website at www.bioinf.org.uk / abs (maintained by AC Martin at the Department of Biochemistry & Molecular Biology University College London, London, England) and the VBASE2 website at www.vbase2.org, as described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005).Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See the chapter "Protein Sequence and Structure Analysis of Antibody Variable Domains" in Dr. Andrew C. Martin's book. Also available in the Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available at bioinforg.uk / abs). The Abysis database website further includes general rules that have been developed for determining CDRs that can be used in accordance with the teachings herein.

[0200] In some specific implementations, the TIGIT binding portion includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0201] (a) VH comprises: HCDR1 as shown in SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2; and HCDR3 as shown in SEQ ID NO: 3; and

[0202] (b) VL includes: LCDR1 as shown in SEQ ID NO: 4; LCDR2 as shown in SEQ ID NO: 5; and LCDR3 as shown in SEQ ID NO: 6.

[0203] In some specific implementations, the PVRIG binding portion includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0204] (a) VH comprises: HCDR1 as shown in SEQ ID NO: 7; HCDR2 as shown in SEQ ID NO: 8; and HCDR3 as shown in SEQ ID NO: 9; and

[0205] (b) VL includes: LCDR1 as shown in SEQ ID NO: 10; LCDR2 as shown in SEQ ID NO: 11; and LCDR3 as shown in SEQ ID NO: 12.

[0206] In some embodiments, the bispecific antibody or its antigen-binding portion includes a first antigen-binding portion that specifically binds to TIGIT, wherein the first antigen-binding portion includes:

[0207] (A) Heavy chain variable region:

[0208] (i) It contains the amino acid sequence of SEQ ID NO: 13;

[0209] (ii) It contains at least 85%, 90%, or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) of the same amino acid sequence as SEQ ID NO: 13; or

[0210] (iii) It comprises an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) added, deleted and / or substituted amino acids compared to SEQ ID NO: 13; and / or

[0211] (B) Light chain variable region:

[0212] (i) It contains the amino acid sequence of SEQ ID NO: 14;

[0213] (ii) It contains at least 85%, at least 90%, or at least 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)) the same amino acid sequence as SEQ ID NO: 14; or

[0214] (iii) It comprises an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) amino acids added, deleted and / or substituted compared to SEQ ID NO: 14.

[0215] In some further embodiments, the bispecific antibody or its antigen-binding portion includes a second antigen-binding portion that specifically binds to PVRIG, wherein the second antigen-binding portion includes:

[0216] (A) Heavy chain variable region:

[0217] (i) It contains the amino acid sequence of SEQ ID NO: 15;

[0218] (ii) It contains at least 85%, 90%, or 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%) of the same amino acid sequence as SEQ ID NO: 15; or

[0219] (iii) It comprises an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) added, deleted and / or substituted amino acids compared to SEQ ID NO: 15; and / or

[0220] (B) Light chain variable region:

[0221] (i) It contains the amino acid sequence of SEQ ID NO: 16;

[0222] (ii) It contains at least 85%, at least 90%, or at least 95% (preferably at least 90%, more preferably at least 95% (e.g., 95%, 96%, 97%, 98%, or 99%)) the same amino acid sequence as SEQ ID NO: 16; or

[0223] (iii) It comprises an amino acid sequence having one or more (e.g., one, two, three or more, preferably one, two or three, more preferably one or two) amino acids added, deleted and / or substituted compared to SEQ ID NO: 16.

[0224] Preferably, the amino acid sequence that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 13, 14, 15, or 16 has the same CDR sequence as SEQ ID NO: 13, 14, 15, or 16, and the amino acid variation occurs only in the frame region. The percentage of identity between the two amino acid sequences can be determined as follows: using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm in the GAP program (available at http: / / www.gcg.com), which is incorporated into the GCG software package, using a Blossum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0225] Alternatively or additionally, the protein sequences disclosed herein can be further used as “query sequences” to search public databases for, for example, to identify relevant sequences. Such searches can be performed using the XBLAST program (version 2.0) as described in Altschul, et al. (1990) J.MoI. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the antibody molecules of this disclosure. For obtaining gapped alignments for comparative purposes, Gapped BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0226] In some specific embodiments, the heavy chain variable region of the TIGIT binding site is composed of the amino acid sequence of SEQ ID NO: 13, and the light chain variable region of the TIGIT binding site is composed of the amino acid sequence of SEQ ID NO: 14, and / or the heavy chain variable region of the PVRIG binding site is composed of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region of the PVRIG binding site is composed of the amino acid sequence of SEQ ID NO: 16.

[0227] In other embodiments, the amino acid sequences of the heavy chain variable region and / or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding sequences shown above, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, and more preferably at least 95%, 96%, 97%, 98%, or 99%.

[0228] In some further embodiments, the bispecific antibody or its antigen-binding moiety may contain conserved substitutions or modifications of amino acids in the variable regions of the heavy and / or light chains. It will be understood in the art that certain conserved sequence modifications can be made without removing the antigen-binding component. See, e.g., Brummell et al. (1993) Biochem 32:1180-8; deWildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley andO' Connell (1993) Biochem.32:6862-35; Adib-Conquy et al. (1998) Int. Immunol.10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0229] As described above, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain. This other amino acid residue is, for example, a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain family. The methods used to determine conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0230] Antigen binding of bispecific polypeptide complexes

[0231] Preferably, the bispecific polypeptide complex of this disclosure is capable of binding to human TIGIT and PVRIG. The binding of the antibody to TIGIT and PVRIG can be assessed using one or more well-established techniques in the art, such as ELISA or FACS, which measure the binding of the antibody to soluble TIGIT / PVRIG proteins or TIGIT / PVRIG proteins expressed on the cell surface, respectively. For example, the antibody can be tested by flow cytometry, wherein the antibody reacts with cell lines expressing human TIGIT or human PVRIG (such as CHO cells transfected to express TIGIT or PVRIG on their cell surface, or TIGIT or PVRIG-positive cell lines, or TIGIT and PVRIG double-positive cell lines).

[0232] Alternatively or concurrently, antibody binding can be tested in a BIAcore binding assay, including binding kinetics (e.g., KD value). For example, the bispecific antibody of this disclosure can be tested at 1 × 10⁻⁶. -10 M or smaller K D 8×10 -11 M or smaller K D 6×10 -11 M or smaller K D 4×10 -11 M or smaller K D Or 2×10 -11 M or smaller K D It binds to human TIGIT protein and can reach 5 × 10 -9 M or smaller K D 4×10 -9 M or smaller K D Or 3×10 -9 M or smaller K D It binds to human PVRIG proteins, as measured by surface plasmon resonance.

[0233] BsAb Functionality

[0234] The bispecific antibodies disclosed herein are characterized by specific functional features or properties. In some embodiments, the antibody has one or more of the following properties:

[0235] (a) Specific binding to TIGIT in humans, cynomolgus monkeys and mice;

[0236] (b) Specific binding to human and cynomolgus monkey PVRIG;

[0237] (c) Can be combined with both TIGIT and PVRIG;

[0238] (d) Effectively blocks the binding of PVRIG to PVRL2 and the binding of TIGIT to PVR;

[0239] (e) It does not have cross-activity with paralog proteins of TIGIT and PVRIG;

[0240] (f) In reporter gene assays, NK cell killing and T cell activation assays, it showed better results than control antibodies (WBP364-BMK1 and WBPT117-BMK1) and even combinations of control antibodies.

[0241] (g) It has good antibody development potential, including thermal stability, solubility, hydrophobicity and stress stability;

[0242] (h) It has an acceptable pharmacokinetic profile in monkeys; and

[0243] (i) It showed significant antitumor efficacy in the CT26 mouse model.

[0244] In some embodiments, the control antibody is a monoclonal antibody, such as a monoclonal anti-TIGIT antibody. In some embodiments, the control antibody is WBP364-BMK1 as shown in Table 1. In some embodiments, the control antibody is a monoclonal anti-PVRIG antibody. In some embodiments, the control antibody is WBPT117-BMK1 as shown in Table 1. In some embodiments, the control antibody is a parent antibody from which bispecific antibodies are derived and constructed. In some embodiments, the control antibody is W3642 and / or WT1175.

[0245] In some embodiments, the bispecific peptide complexes disclosed herein exhibit a higher binding affinity for TIGIT compared to monospecific anti-TIGIT antibodies or other anti-TIGIT / PVRIG bispecific antibodies. In some embodiments, the bispecific peptide complexes disclosed herein exhibit a binding affinity for TIGIT that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of monospecific anti-TIGIT antibodies, as measured by SPR or FACS.

[0246] In some embodiments, the bispecific peptide complexes disclosed herein exhibit a higher or comparable binding affinity for PVRIG compared to monospecific anti-PVRIG antibodies or other anti-TIGIT / PVRIG bispecific antibodies. In some embodiments, the bispecific peptide complexes disclosed herein exhibit a binding affinity for PVRIG that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than that of monospecific anti-PVRIG antibodies or other anti-TIGIT / PVRIG bispecific antibodies, as measured by FACS.

[0247] The anti-TIGIT / PVRIG antibody disclosed herein can inhibit the interaction between TIGIT and its ligand PVR (CD155) and between PVRIG and PVRL2 (CD112). Blocking these signaling pathways can, for example, restore T cells from a dysfunctional state to a functional response to antigen stimulation (e.g., proliferation, cytokine production, target cell killing).

[0248] The ability of an antibody to inhibit the interaction between an antigen and its ligand can be assessed by measuring, for example, whether the physical interaction between TIGIT and PVR is reduced in a binding assay. In some embodiments, the binding assay is a competitive binding assay. This assay can be performed in a variety of forms, such as, but not limited to, ELISA assays, flow cytometry, surface plasmon resonance (SPR) assays (e.g., Biacore™), or BioLayer interferometry (e.g., ForteBio Octet™).

[0249] Multiple blockades of TIGIT, PVRIG, and PD-1 / PD-L1 can reverse immunosuppression. The bispecific antibody disclosed herein has the potential to exhibit synergistic effects with anti-PD-1 agents (e.g., anti-PD-1 antibodies) or anti-PD-L1 agents (e.g., anti-PD-L1 antibodies).

[0250] In some preferred embodiments, the antibodies disclosed herein can be combined with other therapeutic agents, such as anticancer agents, including anticancer antibodies and chemotherapeutic agents. These other therapeutic agents may also be antagonists or inhibitors of T-cell co-inhibitors, agonists of T-cell co-activators, or immunostimulatory cytokines.

[0251] In some embodiments, additional therapeutic agents are antibodies that bind to proteins selected from PD-1 / PD-L1, CD47M, GM-CSF, CSF1R, TLR, RIGI, TAM receptor kinase, NKG2A, NKG2D, GD2, EGFR, PDGFRa, SLAMF7, VEGF, CTLA-4, CD20, cCLB8, KIR, and CD52. In some embodiments, additional therapeutic agents are selected from anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD47M antibodies, anti-CSF1R antibodies, anti-TLR antibodies, anti-RIGI antibodies, anti-TAM receptor kinase antibodies, anti-NKG2A antibodies, anti-CD25 antibodies, anti-NKG2D antibodies, anti-GD2 antibodies, anti-PDGFR-a antibodies, anti-SLAMF7 antibodies, anti-VEGF antibodies, anti-CD20 antibodies, anti-cCLB8 antibodies, anti-KIR antibodies, and anti-CD52 antibodies. In some implementations, additional therapeutic agents are selected from Hul4.18K322A, Hu3F8, dinituximab, olaratumab, elotuzumab, ramucirumab, bevacizumab, rituximab, siltuximab, lirilumab, and alemtuzumab.

[0252] Fc area

[0253] This disclosure provides a polypeptide complex comprising a TIGIT-binding moiety, a PVRIG-binding moiety, and a dimer pair, the dimer pair comprising two dimerizing domains that promote the assembly of at least two polypeptide chains. The dimer pair may comprise, for example, but not limited to, constant regions of immunoglobulins, including, for example, the Fc, CH2, and / or CH3 domains of heavy chain immunoglobulins.

[0254] In some embodiments, the bispecific antibody and its antigen-binding portion provided herein may comprise an immunoglobulin constant region and optionally a hinge region, the immunoglobulin constant region comprising an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region (natural or a variant thereof). In some embodiments, the Fc region is a human IgG1 Fc region, such as a wild-type IgG1 Fc region or an Fc variant. The Fc variant may have at least about 80% homology with the natural sequence Fc region, or at least about 90% homology with it, for example, at least about 95% homology with it. In some embodiments, the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or an Fc variant. In some embodiments, the bispecific polypeptide complexes disclosed herein comprise variant Fc regions having one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA, according to EU designations, as described by Kabat et al.), which alter antibody-dependent cytotoxicity (ADCC) or other effector functions. In some implementations, the Fc region may contain one or more amino acid variations (e.g., insertion, deletion, or substitution), resulting in a modified Fc region that has a modified binding interaction between Fc and FcRn or FcγR.

[0255] In some implementations, the Fc region may contain one or more amino acid variations (e.g., insertion, deletion, or substitution) to achieve the desired functionality, such as a "knob into hole" structure to facilitate heterodimerization (if necessary).

[0256] As used herein, the term "button-in-pore" refers to the engineered CH3 domain of the antibody Fc region to create a "button" or "pore" in each heavy chain to facilitate heterodimerization. A button can be obtained by replacing a small amino acid residue with a larger amino acid residue in the first CH2 / CH3 polypeptide, and a pore can be obtained by replacing a large amino acid residue with a smaller amino acid residue. For details on the mutation sites for button-in-pore, see Spiess et al., 2015, ibid. and Brinkmann et al., 2017, ibid. Typically, according to EU designations, as described by Kabat et al., a "button" is created by replacing T366 with a large residue W on one heavy chain, and the corresponding "pore" is obtained through triple mutations of T366S, L368A, and Y407V on other heavy chains. In some embodiments, the heavy chain containing the bispecific antibody C1 has a "pore" structure, while the heavy chain containing C2 has a "button" structure. Alternatively, the heavy chain of a bispecific antibody containing C1 has a "twist" structure, while the heavy chain containing C2 has a "pore" structure.

[0257] In some embodiments, the Fc region of the bispecific antibody described herein is an IgG4 Fc region containing the S228P mutation (according to EU designations, as described by Kabat et al.), which prevents Fab arm exchange and stabilizes the IgG4 molecule. In some embodiments, the Fc region is an IgG1 Fc region and contains the LALA mutation, namely the mutations of L234A and L235A (according to EU designations, as described by Kabat et al.). The LALA mutation is perhaps the most commonly used mutation for disrupting antibody effector functions (e.g., eliminating Fc binding to specific FcγR, reducing ADCC activity mediated by PBMCs and monocytes).

[0258] In some implementations, the Fc region may be modified to prevent glycosylation, thereby prolonging its half-life to modulate receptor binding or effector function. Exemplary mutations are described in Saunders KO (Front. Immunol. 10:1296, 2019, the entire contents of which are incorporated herein by reference) and include, for example, mutations in asparagine 297 (e.g., N297, according to EU number, as described by Kabat et al.).

[0259] In exemplary embodiments, the polypeptide complexes disclosed herein may have mutated constant regions or Fc regions having a sequence that is 80% to 99% identical to the sequence of the constant regions or Fc regions of natural IgG1, IgG2, IgG3, or IgG4. For example, this document covers mutated Fc regions that are 85% to 99% identical, 90% to 99% identical, or 95% to 99% identical to the sequence of the Fc regions of natural IgG1, IgG2, IgG3, or IgG4.

[0260] When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is commonly used (e.g., the EU index reported by Kabat et al., ibid.). “EU number in Kabat” or “EU index in Kabat” refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody mean residue numbers obtained through the EU numbering system.

[0261] Nucleic acid molecules encoding the antibodies disclosed herein

[0262] In some aspects, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of bispecific antibodies as disclosed herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a heavy chain of a bispecific antibody as disclosed herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a light chain of a bispecific antibody as disclosed herein.

[0263] The nucleic acids disclosed herein can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), the cDNA encoding the light and heavy chains of the antibody prepared via hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0264] By operatively linking the nucleic acid encoding the VH region to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant regions can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are preferably IgG1 or IgG4 constant regions.

[0265] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ constant region or a λ constant region.

[0266] Once the DNA fragments encoding the VH and VL segments are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these operations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used herein, the term "operatively linked" is intended to mean that the two DNA fragments are linked in such a way that the amino acid sequences encoded by the two DNA fragments remain within the frame.

[0267] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding a heavy chain variable region (TIGIT-binding portion or PVRIG-binding portion) or heavy chain encoding a bispecific antibody as disclosed herein.

[0268] In some specific implementations, the isolated nucleic acid molecules include:

[0269] (A) A nucleic acid sequence encoding an amino acid sequence as shown in SEQ ID NO: 13, 15, 17 or 19;

[0270] (B) A nucleic acid sequence that hybridizes to the complementary strand of (A) under highly stringent conditions; or

[0271] (C) is a nucleic acid sequence that has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with (A).

[0272] In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding a light chain variable region (TIGIT-binding portion or PVRIG-binding portion) or a light chain encoding a bispecific antibody as disclosed herein.

[0273] In some specific implementations, the isolated nucleic acid molecules include:

[0274] (A) A nucleic acid sequence encoding the light chain variable region as shown in SEQ ID NO: 14, 16, 18 or 20;

[0275] (B) A nucleic acid sequence that hybridizes to the complementary strand of (A) under highly stringent conditions; or

[0276] (C) is a nucleic acid sequence that has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with (A).

[0277] In some implementations, the identity percentage is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0278] Exemplary high-tightness conditions include hybridization at 45°C in 5X SSPE and 45% formamide, followed by a final wash at 65°C in 0.1X SSC. It will be understood in the art that conditions of equivalent tightness can be achieved by variations in temperature and buffer or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, JohnWiley & Sons (1994), pp. 6.0.3 to 6.4.10. Modifications to the hybridization conditions can be determined empirically or precisely calculated based on the length and percentage of guanosine / cytosine (GC) base pairings of the probe. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold SpringHarbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.

[0279] host cells

[0280] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as yeast, bacteria, fungi, plant and animal cells, such as mammalian cells. Mammalian host cells used for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO cells) (including dhfr CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, which are used with DHFR selection markers, for example, as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), 293F cells, NSO myeloma cells, COS cells, and SP2 cells. Specifically, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841.This also includes monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (for 293 or 293 cell subclones grown in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216); mouse supporting cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL2); and canine kidney cells (MDCK, ATCC CCL 10). 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals NY Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; mouse myeloma cells, such as NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC CRL 1442); 1581); rat myeloma cells, such as YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL 1662); PER.C6 cells; and human hepatocellular carcinoma cell line (Hep G2). CHO cells are one of the cell lines that can be used in this article, of which CHO-K1, DUK-B11, CHO-DP12, CHO-DG44 (Somatic Cell and Molecular Genetics 12:555 (1986)) and Lec13 are exemplary host cell lines. In the case of CHO-K1, DUK-B11, DG44 or CHO-DP12 host cells, these can be altered to lack the ability to fucosylate proteins expressed therein.In some implementations, the host cells used in this paper are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C 6, or NSO cells or lymphocytes.

[0281] Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as those from the family Enterobacteriaceae, including Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacilli (e.g., B. subtilis and B. licheniformis), and Pseudomonas (e.g., Pseudomonas aeruginosa). aeruginosa and Streptomyces.

[0282] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are commonly available and can be used in this paper, such as *Schizo saccharomyces pombe*; hosts of the genus *Kluyveromyces* such as, for example, *Kluyveromyces lactis*, *Kluyveromyces fragilis* (ATCC 12,424), *Kluyveromyces bulgaricus* (ATCC 16,045), *Kluyveromyces wickeramii* (ATCC 24,178), *Kluyveromyces waltii* (ATCC 56,500), *Kluyveromyces drosophilarum* (ATCC 36,906), *Kluyveromyces thermotolerans*, and *Kluyveromyces marxianus*; and *Yarrowia* (EP... 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger.

[0283] When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a sufficient period of time to allow the antibody to be expressed in the host cells or by secreting the antibody into the culture medium in which the host cells are growing. The antibody can be recovered from the culture medium using standard protein purification methods.

[0284] Pharmaceutical Composition

[0285] In some aspects, this disclosure relates to pharmaceutical compositions comprising at least one antibody or its antigen-binding moiety as disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising nucleic acid (DNA or RNA) encoding an antibody as disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, this disclosure provides pharmaceutical compositions comprising cells expressing an antibody as disclosed herein, and a pharmaceutically acceptable carrier.

[0286] Components of the composition

[0287] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions disclosed herein may also be administered in combination therapy with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, and multiple or more combinations of other components known in the art.

[0288] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl gallate. For example, compositions containing antibodies or antigen-binding fragments of the present disclosure may include one or more antioxidants such as methionine, and reducing antibodies or antigen-binding fragments thereof may be oxidized. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or antigen-binding fragments thereof and one or more antioxidants such as methionine. This disclosure further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding fragment to extend its shelf life and / or increase its activity.

[0289] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous media such as sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous media such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents at antibacterial or antifungal concentrations; isotonic agents such as sodium chloride or dextran; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); isolating or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethanol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents, which can be used as carriers, can be added to pharmaceutical compositions in multi-dose containers. These antimicrobial agents include phenols or cresols, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Suitable excipients may include, for example, water, saline, dextran, glycerol, or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers, or reagents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0290] Application, preparation and dosage

[0291] The pharmaceutical compositions disclosed herein can be administered to subjects in need via a variety of routes of administration, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous, and intrathecal administration, or otherwise via implantation or inhalation. The subject compositions can be formulated into solid, semi-solid, liquid, or gaseous forms; these include, but are not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0292] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets (including coated tablets), elixirs, suspensions, syrups, or inhalers, and their controlled-release forms.

[0293] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). Such liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of suitable isotonic carriers for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, a particular dosing regimen, including dosage, time, and repetition, will depend on the individual and that individual's medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance, etc.).

[0294] The frequency of administration can be determined and adjusted during the course of therapy, and is based on reducing the number of proliferating or tumorigenic cells, maintaining the reduction of such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dosage may be adjusted or reduced to manage potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the subject therapeutic composition may be suitable.

[0295] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage will generally involve balancing the level of therapeutic benefit against any risks or harmful side effects. The chosen dosage level will depend on a variety of factors, including, but not limited to, the activity of the particular compound, route of administration, time of administration, rate of excretion of the compound, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and the patient's species, sex, age, weight, condition, general health, and medical history. The amount of compound and route of administration will ultimately be determined by the physician, veterinarian, or clinician, although a dosage will generally be chosen to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or toxic side effects.

[0296] Typically, the antibodies or antigen-binding portions thereof disclosed herein can be administered in a variety of ranges. These include about 5 μg / kg body weight to about 40 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; and about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0297] In summary, the antibodies or antigen-binding portions thereof disclosed herein are preferably administered to subjects as needed. The frequency of administration can be determined by those skilled in the art, such as by an attending physician based on considerations such as the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health status of the subject being treated.

[0298] In some preferred embodiments, a treatment procedure involving the antibody or its antigen-binding portion of this disclosure will comprise a selection of multiple doses of the pharmaceutical product over a period of weeks or months. More specifically, the antibody or its antigen-binding portion of this disclosure may be administered once daily, every two days, every four days, once weekly, every ten days, every two weeks, every three weeks, once monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it should be understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0299] The dosage and regimen of the disclosed therapeutic composition can also be determined empirically in individuals who have been given one or more administrations. For example, individuals can be given incremental doses of the therapeutic composition produced as described herein. In selected embodiments, the dosage can be gradually increased, decreased, or weakened based on empirically determined or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, symptom, or condition can be tracked as previously described. For cancer, these include direct measurement of tumor size via palpation or visual observation, indirect measurement of tumor size via X-ray or other imaging techniques; improvement assessed as such by direct tumor biopsy and microscopic examination of tumor samples; measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens identified according to the methods described herein; reduction of pain or paralysis; improvement in tumor-associated speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or prolonged survival as measured by recognized tests.

[0300] In some embodiments, the compatible formulation for parenteral administration (e.g., intravenous injection) comprises an antibody or its antigen-binding portion as disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. It will be apparent to those skilled in the art that the dosage of the antibody or its antigen-binding portion as disclosed herein can vary depending on the individual, the type of neoplastic condition, the stage of the neoplastic condition, whether the neoplastic condition has begun to metastasize to other sites in the individual, past and concurrent treatments being used, and the dosage of the therapeutic agent used in combination with the antibody as disclosed herein.

[0301] Application of this disclosure

[0302] The antibodies, antibody compositions, and methods disclosed herein have numerous in vitro and in vivo utilities, relating to, for example, the detection of TIGIT / PVRIG or the enhancement of immune responses. For example, these molecules can be administered in vitro or ex vivo to cells in cultures, or, for example, in vivo to human subjects, to enhance immunity in a variety of situations. Immune responses can be modulated, for example, enhanced, stimulated, or upregulated.

[0303] For example, subjects include patients who require enhanced immune responses. This method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses (e.g., T-cell-mediated immune responses). In certain embodiments, this method is particularly suitable for treating cancer in vivo. When the bispecific antibody is administered together with another agent, such as an anti-PD-L1 agent, the two can be administered in either order or simultaneously.

[0304] Treatment of diseases including cancer

[0305] In some aspects, this disclosure provides methods for treating conditions or diseases in mammals, comprising administering to a subject (e.g., a human) a therapeutically effective amount of a bispecific antibody or its antigen-binding portion disclosed herein, for example, in combination with a PD-1 / PD-L1 antagonist. Conditions or diseases include, but are not limited to, proliferative conditions (such as cancer), immune conditions, inflammatory diseases, or infectious diseases. For example, a condition may be cancer.

[0306] In some embodiments, the cancer is cancer rich in the expression of CD112, CD113, or CD155. In some embodiments, the cancer is cancer rich in T cells or natural killer (NK) cells expressing TIGIT. In some embodiments, the cancer is cancer rich in the expression of PRVL2. In some embodiments, the cancer is cancer rich in T cells or natural killer (NK) cells expressing PVRIG.

[0307] Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, lung cancer, such as non-small cell lung cancer (NSCLC), which includes squamous or non-squamous NSCLC, including locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC), or recurrent or metastatic NSCLC (e.g., stage IV NSCLC); lung adenocarcinoma or squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer. Cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-refractory non-muscle-invasive bladder cancer (NMIBC)); urethral cancer; liver cancer; breast cancer (e.g., TIGIT+ breast cancer and triple-negative breast cancer (TNBC), which is estrogen receptor (ER-), progesterone receptor (PR-), and TIGIT (TIGIT-) negative); colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary gland cancer; kidney or renal cancer (e.g., renal cell carcinoma (RCC)); prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma, including superficial diffuse melanoma and malignant lentigo maligna melanoma. Melanoma, acral lentigines melanoma and nodular melanoma; multiple myeloma and B-cell lymphoma (including low-grade / follicular non-Hodgkin lymphoma (NHL)); small lymphocytic (SL) NHL; intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD);And myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as edema associated with brain tumors), Megs syndrome, brain cancer, head and neck cancer, and related metastases.

[0308] As a co-inhibitory receptor on a variety of immune cells, TIGIT or PVRIG is involved in a variety of cancers (whether malignant or benign, and whether primary or secondary), which can be treated or prevented by the methods provided in this disclosure. The cancer can be a solid tumor or a hematologic malignancy. Examples of such cancers include lung cancers such as bronchogenic carcinomas (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromatous hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyosarcoma; bone cancers such as osteochondroma, condromas, chondroblastoma, chondromycinoid fibroma, osteoid osteoma, giant cell tumors, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; and brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytomas, and other neurological disorders. Cancers of the digestive system, including: astrocytomas, oligodendrogliomas, medulloblastomas, chordomas, schwannomas, ependymomas, meningiomas, pituitary adenomas, pineal tumors, osteomas, angioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts, and hemangiomas; cancers of the digestive system, such as colon cancer, leiomyomas, epidermoid carcinomas, adenocarcinomas, leiomyosarcomas, gastric adenocarcinomas, intestinal lipomas, enterofibromas, intestinal fibromas, colorectal polyps, and colorectal cancer; liver cancers, such as hepatocellular adenomas, hemangiomas, hepatocellular carcinomas, fibrolamellar carcinomas, cholangiocarcinomas, hepatoblastomas, and angiosarcomas; and kidney cancers, such as renal adenocarcinomas, renal cell carcinomas, adrenoid adenomas, and renal pelvis tumors. Transitional cell carcinoma; bladder cancer; skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancer; eye-related cancers such as retinoblastoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid gland; pancreatic cancer. In a particular implementation, the cancer is colon cancer.

[0309] In some other embodiments, the condition or disease to be treated or prevented is an immune-related disease. Immune-related diseases may be associated with T-cell dysfunction. In some embodiments, T-cell dysfunction is characterized by reduced responsiveness to antigen stimulation. In some embodiments, T-cell dysfunction is characterized by T-cell incompetence, or reduced ability to secrete cytokines, proliferate, or perform cytolytic activities. In some embodiments, T-cell dysfunction is characterized by T-cell exhaustion. In some embodiments, the T cells are CD4+ T cells and CD8+ T cells. In some embodiments, immune-related diseases are selected from the group consisting of unresolved acute infections, chronic infections, and reduced tumor immunity.

[0310] Stimulation of immune response

[0311] In some aspects, this disclosure also provides methods for enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject an antibody of this disclosure or an antigen-binding portion thereof, such that an immune response in the subject is enhanced. For example, the subject is a mammal. In a particular embodiment, the subject is a human.

[0312] The term "enhanced immune response" or its grammatical variations refer to any response that stimulates, evokes, increases, improves, or strengthens the immune system of a mammal. An immune response can be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., an antibody-mediated response), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+. + This method enhances the activity of helper T cells and the generation of cytolytic T cells. Enhancement of the immune response can be assessed using many in vitro or in vivo measurements known to those skilled in the art, including but not limited to, cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-bearing animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Generally, the methods of this disclosure enhance the immune response of mammals when compared to the immune response of untreated mammals or mammals not treated with methods as disclosed herein. In one embodiment, the antibody or its antigen-binding portion is used to enhance the human immune response to microbial pathogens (such as viruses). In another embodiment, the antibody or its antigen-binding portion is used to enhance the human immune response to a vaccine. In one embodiment, the method enhances cellular immune responses, particularly cytotoxic T cell responses. In another embodiment, the cellular immune response is a T helper cell response. In yet another embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-2 production. The antibody or its antigen-binding portion can be used to enhance the human immune response to microbial pathogens (such as viruses) or to a vaccine.

[0313] Antibodies or their antigen-binding portions can be used alone as a monotherapy or in combination with chemotherapy, radiotherapy, targeted therapy or cell immunotherapy.

[0314] Used in combination with chemotherapy

[0315] Antibodies or their antigen-binding portions can be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0316] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat proliferative disorders such as cancer, and include, but are not limited to, cytotoxic agents, cytostatic agents, anti-angiogenic agents, debulking agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and antimetastatic agents, and immunotherapy agents. It should be understood that such anticancer agents may comprise conjugates and may be associated with the disclosed antibody prior to administration. More specifically, in some embodiments, the selected anticancer agent will be linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate. Therefore, such engineered conjugates are explicitly considered to be within the scope of this disclosure. In some other embodiments, the anticancer agent will be administered in combination with an antibody-drug conjugate comprising a different therapeutic agent.

[0317] As used herein, the term "cytotoxic agent" means a substance that is toxic to cells and reduces or inhibits cellular function and / or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, the following small molecule toxins or enzymatically active toxins: bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-ascorbic acid toxin, localized arbutin), plants (e.g., absinthecin, ricin, senna root toxin, viscumin, pokeweed antiviral protein, saponins, white arbutin, momoridin, trichosanthes pollen protein, barley toxin, tung oil (Aleuritesfordii) protein, caryophyllin protein, Phytolacca mericana protein (PAPI, PAPII, and PAP-S), bitter melon (Momordica) Inhibitors of charantia, jatropha toxin, croton toxin, saponaria officinalis inhibitor, white tree toxin, mitegellin, localized aspergillin, phenolmycin, neomycin and trichothecene toxins) or animals (e.g., cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including its fragments and / or variants).

[0318] For the purposes of this disclosure, "chemotherapeutic agents" comprise chemical compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents typically target intracellular processes necessary for cell growth or division, and are therefore particularly effective against cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or generate tumorigenic progeny (e.g., TIC). Such agents are often administered in combination and are often the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0319] Examples of anticancer agents that can be used in combination with the bispecific antibodies of this disclosure (either as a component of a site-specific conjugate or in an unconjugated state) include, but are not limited to, alkylating agents, alkylsulfonates, aziridines, ethylenimines and methylamelamines, acetogenins, camptothecin, bryostatin, callystatin, CC-1065, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esperamicin, and chromoprotein enediyne antibiotic chromophores. Antibiotic chromophores, aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin C, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN ®Doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;Anti-metabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogues, purine analogues, androgens, anti-adreners, folic acid supplements such as frolinic acid, aceglatone, aldophosphamide glycoside, and aminolevulinic acid. (acid), eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate nitrate), hydroxyurea, lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllinicacid, 2-ethylhydrazide, procarbazine, PSK; ®Polysaccharide complexes (JHS Natural Products, Eugene, OR), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactalol; piperobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids; chloranbucil; GEMZAR ® Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Navelbine ®Vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine; retinoids; capecitabine; compretastatin; leucovorin; oxaliplatin; inhibitors of PKC-α, Raf, and VEGF-A that inhibit cell proliferation; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents that regulate or inhibit the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit the enzyme aromatase (which regulates estrogen production in the adrenal glands), and anti-androgens; as well as troxatabine (a 1,3-dioxolane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors; vaccines, PROLEUKIN ® rIL-2; LURTOTECAN ® Topoisomerase 1 inhibitor; ABARELIX ® rmRH; vinorelbine and esperamycin and any of the above pharmaceutically acceptable salts, acids or derivatives.

[0320] Used in combination with radiotherapy

[0321] This disclosure also provides combinations of antibodies or their antigen-binding portions with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma radiation, X-rays, UV radiation, microwaves, electron emission, etc.). Combination therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed antibodies can be used in combination with targeted anticancer agents or other targeted approaches. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy can be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy can be administered as a single dose or as multiple consecutive doses.

[0322] Drug packaging and reagent kits

[0323] Pharmaceutical packages and kits comprising one or more containers are also provided, containing one or more doses of an antibody or its antigen-binding portion. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding portion, with or without one or more additional pharmaceutical agents. In other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe for injection. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, etc.; buffers, such as phosphates, etc.; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the encapsulated antibody is intended for the treatment of a selected oncological condition.

[0324] This disclosure also provides kits containing a single or multiple dose unit of antibody and optionally one or more anticancer agents. The kit includes a container and a label or packaging insert on or associated with the container. Suitable containers include bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic and contains a pharmaceutically effective amount of the disclosed antibody. In some embodiments, the container includes a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be penetrated by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally contain one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding portion disclosed herein, such kits may contain any one or more of a range of anticancer agents, such as chemotherapeutic or radiotherapy agents; antiangiogenic agents; antimetastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents. In some embodiments, the kit may contain an antiPD-1 antibody.

[0325] More specifically, the kit may have a single container containing an antibody or its antigen-binding moiety, with or without additional components, or they may have different containers for each desired agent. In cases where combination therapies are provided for conjugation, single solutions may be premixed in molar equivalents or in a manner where one component predominates over another. Alternatively, the antibody and any optional anticancer agent in the kit may be maintained separately in different containers prior to administration to the patient. The kit may also include a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as antibacterial water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextran solution.

[0326] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0327] As briefly noted above, the kit may also contain means by which to administer the antibody or its antigen-binding portion and any optional components to a patient, such as one or more needles, IV bags, or syringes, or even droppers, pipettes, or other similar devices from which the preparation may be injected or introduced into an animal or applied to a diseased area of ​​the body. The kits disclosed herein will also typically include means for containing vials, etc., and other tightly sealed components for commercial sale, such as injection or blow-molded plastic containers in which the desired vials and other equipment are placed and held.

[0328] Summary of sequence lists

[0329] Attached to this application is a sequence listing containing numerous amino acid sequences. Tables A, B, C, and D below provide a summary of the included sequences. The CDRs for the anti-TIGIT moiety or anti-TIGIT antibody are determined according to the Contact definition scheme, while the CDRs for the anti-PVRIG moiety or anti-PVRIG antibody are determined according to the IMGT / Kabat definition scheme (HCDR1 determined by the combination of the IMGT and Kabat schemes; other CDRs determined by the Kabat scheme). W3XX104-T4U1.G15-2.uIgG1 and W3XX104-T4U1.G17-2.uIgG1 are bispecific antibodies constructed from parental antibodies W3642 (anti-TIGIT) and WT1175 (anti-PVRIG).

[0330] Table A: CDR sequences of bispecific antibodies W3XX104-T4U1.G15-2.uIgG1 and W3XX104-T4U1.G17-2.uIgG1

[0331]

[0332] Table B: Amino acid sequence of the variable region of BsAb (CDR underlined)

[0333]

[0334] Table C: Sequences of the heavy and light chains of BsAb (CDR with underlined lines)

[0335]

[0336] Table D: Antigen Sequence

[0337]

[0338] Example

[0339] The present disclosure, as generally described herein, will be more readily understood by referring to the following embodiments, which are provided by way of illustration and are not intended to limit the present disclosure. The embodiments are not intended to represent all or only the experiments conducted.

[0340] Example 1

[0341] Preparation of antigens, reference antibodies and cell lines

[0342] 1.1 Antigen generation

[0343] W364-hPro1.ECD.His is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal multihistidine tag; W364-hPro1.ECD.hFc is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal Fc region containing human IgG1; W364-mPro1.ECD.His is the extracellular domain of mouse TIGIT (NP_001139797.1) with a C-terminal multihistidine tag; W364-mPro1.ECD.hFc is the extracellular domain of mouse TIGIT (NP_001139797.1) with a C-terminal Fc region containing human IgG1; W364-hPro1L1.ECD.hFc is the extracellular domain of human CD155 (NP_006496.3) with a C-terminal Fc region containing human IgG1. W364-hPro1L1.ECD.mFc is the extracellular domain of human CD155 (NP_006496.3) with a mouse IgG1 Fc region at the C-terminus.

[0344] WT117-hPro1.ECD.His is the extracellular domain of a human PVRIG (NP_076975.2) with a C-terminal multihistidine tag; WT117-hPro1.ECD.hFc is the extracellular domain of a human PVRIG (NP_076975.2) with a C-terminal Fc region containing human IgG1; WT117-hPro1.ECD.mFc is the extracellular domain of a human PVRIG (NP_076975.2) with a C-terminal Fc region containing mouse IgG2a; WT117-mPro1.ECD.His is the extracellular domain of a mouse PVRIG (XP_011239268.1) with a C-terminal multihistidine tag; WT117-mPro1.ECD.hFc is the extracellular domain of a mouse PVRIG with a C-terminal Fc region containing human IgG1. The extracellular domain of (XP_011239268.1); WT117-hPro1L1.ECD.mFc is the extracellular domain of human PVRL2 (NP_001036189.1) with a mouse IgG2a Fc region at the C-terminus.

[0345] These antigens are purchased from suppliers or prepared in-house.

[0346] 1.2 Preparation of the reference antibody (BMK)

[0347] In the following experiments, anti-human TIGIT or PVRIG reference antibodies, named WBP364-BMK1 and WBPT117-BMK1 in this paper, were used as controls. The sequences of these two antibodies were synthesized based on the sequences disclosed in their respective patents, and their information is summarized in Table 1. The human IgG1 isotype control antibody is an isotype control.

[0348] Table 1. Reference Antibody Information

[0349]

[0350] 1.3 Cell Pool / Cell Line Generation

[0351] The human TIGIT-expressing cell line W364-CHOK1.hPro1.2A11 was generated using CHOK1 cells transfected with full-length human TIGIT (NP_776160.2). The cynomolgus monkey TIGIT-expressing cell pool W364-FlpinCHO.cynoPro1.pool was generated using FlpinCHO cells transfected with full-length cynomolgus monkey TIGIT (XP_015300911.1). The mouse TIGIT-expressing cell pool W364-FlpinCHO.mPro1.pool was generated using FlpinCHO cells transfected with full-length mouse TIGIT (NP_001139797.1).

[0352] The human PVRIG-expressing cell line WT117-293F.hPro1.G11 was generated using 293F cells transfected with full-length human PVRIG (NP_076975.2). The cynomolgus monkey PVRIG-expressing cell pool WT117-Flpin293.cPro1.pool was generated using Flpin293 cells transfected with full-length cynomolgus monkey PVRIG (XP_005549281.1). The mouse PVRIG-expressing cell pool WT117-293F.mPro1.pool was generated using 293F cells transfected with full-length mouse PVRIG (XP_011239268.1).

[0353] Example 2

[0354] Generation of bispecific antibodies

[0355] 2.1 Plasmid Construction

[0356] Monospecific anti-PVRIG and anti-TIGIT antibodies were developed. The anti-TIGIT monoclonal antibody W3642 (or "T4") was obtained by immunizing Sprague-Dawley (SD) rats and then humanizing them, and the anti-PVRIG monoclonal antibody WT1175 (or "U1") was obtained by immunizing genetically engineered OmniRat. Their variable regions were extracted for the construction of bispecific anti-PVRIG / TIGIT antibodies.

[0357] Construction of the G15 form of bispecific antibody: The DNA sequence encoding the anti-PVRIG antibody scFv (VH-(G4S)4-VL) was linked to the C-terminus of the full-length heavy chain of the anti-TIGIT antibody using a (G4S)4 linker, resulting in the form IgG (H)-scFv. The sequence was cloned into the pcDNA3.4 expression vector. The obtained bispecific antibody was named “W3XX104-T4U1.G15-2.uIgG1”, “T4U1.G15”, or “G15”. A schematic diagram of the structure of W3XX104-T4U1.G15-2.uIgG1 is shown below. Figure 1 The image on the left.

[0358] Construction of the G17 bispecific antibody: The DNA sequence encoding the anti-TIGIT antibody scFv (VH-(G4S)4-VL) was linked to the N-terminus of the full-length heavy chain of the anti-PVRIG antibody using a (G4S)4 linker, resulting in the form scFv-IgG (H). The sequence was cloned into the pcDNA3.4 expression vector. The obtained bispecific antibody was named “W3XX104-T4U1.G17-2.uIgG1”, “T4U1.G17”, or “G17”. A schematic diagram of the structure of W3XX104-T4U1.G17-2.uIgG1 is shown below. Figure 1 The image on the right.

[0359] The sequences of the above antibodies are summarized in Table AC above.

[0360] 2.2 Expression in Expi293 cells

[0361] Expi293 cells (Thermo Fisher, catalog number A14635) were used for protein expression. Plasmids containing the heavy and light chains of bispecific antibodies were transfected into Expi293 cells. Following transfection, the culture was incubated at 37°C with 8% CO2 for 5 days until the supernatant was harvested for protein purification.

[0362] 2.3 Antibody purification

[0363] Supernatant from Expi293 cell cultures was collected and filtered for purification using a Protein A column (GE Healthcare, catalog number 175438). The concentration of the purified antibody was determined by UV absorbance at 280 nm. Molecular weight and purity were determined by SDS-PAGE and SEC-HPLC, respectively.

[0364] Example 3

[0365] In vitro characterization

[0366] 3.1 Human TIGIT or PVRIG binding assay

[0367] W364-CHOK1.hPro1.2A11 or WT117-293F.hPro1.G11 cells were incubated with different concentrations of antibody at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, PE-labeled goat anti-human IgG (Jackson Immuno Research, catalog number 109-115-098) was added, and the cells were incubated in the dark at 4°C for 1 hour. Anti-human TIGIT antibody WBP364-BMK1 and anti-PVRIG antibody WBPT117-BMK1 were used as their respective positive controls. Human IgG1 isotype control antibody was used as an isotype control. The cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0368] The binding results of the antibodies to W364-CHOK1.hPro1.2A11 or WT117-293F.hPro1.G11 showed that... Figure 2 and Figure 3 These results indicate that both G17 and G15 bind to human TIGIT-expressing cells W364-CHOK1.hPro1.2A11 with potency comparable to WBP364-BMK1, and both G17 and G15 bind to PVRIG-expressing cells WT117-293F.hPro1.G11 with greater potency than WBPT117-BMK1. A summary of antibody binding is shown in Table 4.

[0369] 3.2 Binding assay of TIGIT or PVRIG in cynomolgus monkeys

[0370] W364-FlpinCHO.cynoPro1.pool or WT117-Flpin293F.cPro1.pool cells were incubated with different concentrations of antibody at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, PE-labeled goat anti-human IgG (Jackson ImmunoReasearch, catalog number 109-115-098) was added as a secondary antibody, and the cells were incubated in the dark at 4°C for 1 hour. Anti-human TIGIT antibody WBP364-BMK1 and anti-PVRIG antibody WBPT117-BMK1 were used as their respective positive controls. Human IgG1 isotype control antibody was used as an isotype control. Cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0371] The binding results of anti-TIGIT / PVRIG BsAb to W364-FlpinCHO.cynoPro1.pool or WT117-Flpin293F.cPro1.pool showed... Figure 4 and Figure 5 These results indicate that both G17 and G15 bind to cynomolgus monkey TIGIT-expressing cells (W364-FlpinCHO.cynoPro1.pool) with potency comparable to WBP364-BMK1, and both G17 and G15 bind to PVRIG-expressing cells (WT117-Flpin293F.cPro1.pool) with greater potency than WBPT117-BMK1. A summary of antibody binding is shown in Table 4.

[0372] 3.3 Mouse TIGIT Binding Assay

[0373] W364-FlpinCHO.mPro1.pool cells were incubated with different concentrations of antibody at 4°C for 1 hour. After washing with 1xPBS / 1% BSA, PE-labeled goat anti-human IgG (Jackson ImmunoReasearch, catalog number 109-115-098) was added, and the cells were incubated in the dark at 4°C for 1 hour. Human IgG1 isotype control antibody was used as an isotype control. The cells were then washed and resuspended in 1xPBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0374] The binding results of anti-TIGIT / PVRIG BsAb to W364-FlpinCHO.mPro1.pool cells showed that Figure 6 The results showed that both G17 and G15 bound to cells expressing mouse TIGIT, while WBP364-BMK1 did not. A summary of antibody binding is shown in Table 4.

[0375] 3.4 Mouse PVRIG Binding Assay

[0376] WT117-293F.mPro1.pool cells were incubated with different concentrations of antibody at 4°C for 1 hour. After washing with 1xPBS / 1%BSA, PE-labeled goat anti-human IgG (Jackson ImmunoReasearch, catalog number 109-115-098) was added as a secondary antibody, and the cells were incubated in the dark at 4°C for 1 hour. Anti-mouse PVRIG antibody fused with human IgG1 Fc was used as a positive control. Human IgG1 isotype control antibody was used as an isotype control. The cells were then washed and resuspended in 1xPBS / 1%BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0377] The binding results of anti-TIGIT / PVRIG BsAb to WT117-293F.mPro1.pool cells showed that Figure 7 The results showed that neither G17 nor G15, nor WBPT117-BMK1, bound to mouse PVRIG.

[0378] 3.5 Human TIGIT or PVRIG binding affinity assay

[0379] The affinity of anti-TIGIT / PVRIGBsAbs for recombinant human TIGIT or PVRIG was determined using a Biacore 8K instrument (Cytiva) via surface plasmon resonance (SPR). Goat anti-human IgG Fc antibody (Jackson ImmunoResearch, catalog number 109-005-098) was immobilized on a CM5 biosensor chip (GE, catalog number 29-1496-03), and the antibody was tested by goat anti-human IgG Fc antibody capture. For kinetic measurements, a series of concentrations of W364-hPro1.ECD.His or WT1175-hPro1.ECD.His were injected at 25 °C at a flow rate of 30 μL / min into running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) for a binding phase, followed by a dissociation phase. The binding rate (kon) and dissociation rate (koff) were calculated using a simple one-to-one Languir binding model. The equilibrium dissociation constant (KD) was calculated as the koff / kon ratio.

[0380] The SPR binding affinity results of anti-TIGIT / PVRIG BsAb to human TIGIT or PVRIG showed that... Figure 2 and Figure 3In the assay, G17 bound to human TIGIT and PVRIG proteins with affinities of 1.17E-11 and 2.69E-09 M, respectively. G15 bound to human TIGIT and PVRIG proteins with affinities of 1.87E-11 and 5.42E-09 M, respectively. A summary of antibody binding affinities is shown in Table 4, and the sensor plot is shown in... Figure 8 and Figure 9 middle.

[0381] Table 2. Affinity constants of antibodies to human TIGIT

[0382]

[0383] Table 3. Affinity constants of antibodies to human PVRIG

[0384]

[0385] 3.6 Paralog Protein Binding Assay

[0386] The plate was pre-coated overnight at 4°C with 1 μg / mL of W364-hPro1.ECD.His, WT117-hPro1.ECD.His, recombinant human CD226, CD96, or PD-1 extracellular domain in 100 μL of coating buffer (Na2CO3 / NaHCO3, pH 9.2) per well. After blocking with 200 μL of 1xPBS / 2% BSA, 100 μL of the test antibody at a concentration of 66.67 nM was added to the plate, and the plate was incubated at ambient temperature for 1 hour. After incubation, the plate was washed three times with 1xPBST. HRP-labeled goat anti-human IgG antibody (Bethyl, catalog number A80-304P) diluted in 1xPBS / 2% BSA was added, and the plate was incubated at ambient temperature for 1 hour. After washing 6 times with 1xPBST, color development was performed by dispensing 100 μL of TMB substrate, and then the reaction was stopped by adding 100 μL of 2M HCl. The absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0387] The binding results of anti-TIGIT / PVRIG BsAb to TIGIT and PVRIG paralogous proteins showed that... Figure 10 The results showed that both G17 and G15 specifically bind to TIGIT and PVRIG, but have no cross-reactivity with human CD226, CD96, and PD-1.

[0388] 3.7 Human TIGIT or PVRIG Dual Binding Assay

[0389] The plates were pre-coated with 0.1 μg / mL W364-hPro1.ECD.His in 100 μL of coating buffer per well and incubated overnight at 4°C. After blocking with 200 μL of 1xPBS / 2% BSA, 100 μL of test antibody at different concentrations was added to the plates and incubated at ambient temperature for 2 hours. After incubation, the plates were washed three times with 1xPBST. WT117-hPro1.ECD.mFc was added to the plates at a concentration of 0.1 μg / mL and diluted in 1xPBS / 2% BSA. After incubation at ambient temperature for 1 hour, the plates were washed three times with 1xPBST. HRP-labeled goat anti-mouse IgG antibody (Bethyl, catalog number A90-231P) diluted in 1xPBS / 2% BSA was added and incubated at ambient temperature for 1 hour. After washing six times with 1xPBST, color development was performed by dispensing 100 μl of TMB substrate, followed by stopping the reaction by adding 100 μl of 2M HCl. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0390] The results of dual binding of anti-TIGIT / PVRIG BsAb with fixed human TIGIT and soluble human PVRIG showed that... Figure 11 The results showed that both G17 and G15 bound to both human TIGIT and PVRIG simultaneously, while the reference mAbs WBP364-BMK1 and WBPT117-BMK1 did not bind to both TIGIT and PVRIG. A summary of antibody dual binding is shown in Table 4.

[0391] 3.8 TIGIT / PVR Blockade Measurement

[0392] The ability of anti-TIGIT / PVRIG BsAb to block human TIGIT / PVR interaction was tested by FACS. W364-CHOK1.hPro1.2A11 cells were washed with 1xPBS / 1% BSA and then subjected to 1x10⁻¹¹ PBS / 1% BSA. 5Cells were seeded per well in 96-well round-bottom plates. Excess buffer was removed from the wells by centrifugation. Serially diluted antibody (2x concentration) was premixed with 4 μg / mL (2x concentration) W364-hPro1L1.ECD.mFc at a 1:1 volume ratio, and 100 μL of the antibody / ligand mixture was added to each well. The plate was incubated at 4°C for 1 hour. After washing with 1xPBS / 1%BSA, PE-labeled goat anti-mouse IgG (Bethyl, catalog number A90-239PE) was added, and the cells were incubated with the secondary antibody at 4°C for 1 hour in the dark. Anti-human TIGIT antibody WBP364-BMK1 was used as a positive control. Human IgG1 isotype antibody was used as an isotype control. Cells were then washed and resuspended in 1xPBS / 1%BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed by FlowJo.

[0393] Human TIGIT / PVR blocking results showed Figure 12 The results showed that both G17 and G15 blocked the binding of human PVR to human TIGIT, and they exhibited equivalent blocking efficacy compared to the reference TIGIT mAb WBP364-BMK1. A summary of the antibody blocking activities is shown in Table 4. The maximum inhibition rate was calculated as inhibition% = (MFI) 最大 – MFI 底部 ) / MFI 最大 x 100%, of which MFI 最大 MFI is defined as the absence of antibodies.

[0394] 3.9 Human PVRIG / PVRL2 Blockade Assay

[0395] The ability of anti-TIGIT / PVRIG BsAb to block human PVRIG / PVRL2 interaction was tested by ELISA. Plates were pre-coated overnight at 4°C with 0.5 μg / mL rabbit anti-his antibody in 100 μL of coating buffer per well. After blocking with 200 μL of 1xPBS / 2% BSA, 100 μL of WT117-hPro1.ECD.His at a concentration of 0.8 μg / mL was added to each well and incubated at ambient temperature for 1 hour. Serially diluted antibody (2x concentration) was mixed with 0.6 μg / mL (2x concentration) of WT1175-hPro1L1.ECD.mFc at a 1:1 volume ratio. After incubation, 100 μL of the antibody / ligand mixture was added to the plate and incubated at ambient temperature for 2 hours. After washing three times with 1xPBST, HRP-labeled goat anti-mouse IgG (Bethyl, catalog number A90-231P) was added to the plate and incubated at ambient temperature for 1 hour. After washing six times with 1xPBST, TMB substrate was added, and the interaction was then terminated with 2M HCl. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices).

[0396] Human PVRIG / PVRL2 blocking results showed Figure 13 The results showed that both G17 and G15 blocked the binding of human PVRL2 to human PVRIG, and they exhibited equivalent blocking efficacy compared to the reference PVRIG mAb WBPT117-BMK1. A summary of the antibody blocking activities is shown in Table 4. The maximum inhibition rate was calculated as inhibition% = (OD0.05)2. 最大 – OD 底部 ) / OD 最大 x100%, where OD 最大 It is defined as the value of OD450 minus OD540 in the absence of antibodies.

[0397] 3.10 Jurkat TIGIT / PVRIG / NFAT-luciferase reporter gene assay (RGA)

[0398] Jurkat cells overexpressing human TIGIT, PVRIG, and NFAT-luciferase reporter were stimulated via T-cell receptor binding by co-culturing with artificial APCs. The artificial APCs were engineered HT1080 cells constitutively expressing PVR and PVRL2 and transfected to express a human TCR activator. HT1080 / TCR activator cells were cultured at 2 x 10⁻⁶ cells / year. 4Cells were seeded at a density of 10 cells / well in 96-well plates and incubated overnight at 37°C, 5% CO2. The next day, after removing the supernatant and non-adherent cells, serially diluted antibody and Jurkat / TIGIT / PVRIG / NFAT-luciferase cells (4 x 10⁻⁶ cells / well) were seeded. 4 Cells per well were added to the plate and co-incubated at 37°C, 5% CO2 for 5.5 hours. After incubation, the reconstituted luciferase substrate (Promega, catalog number E605B) was added to each well and mixed thoroughly. Luciferase intensity was read using an Envision microplate reader (PerkinElmer).

[0399] The results of the Jurkat TIGIT / PVRIG / NFAT-luciferase reporter gene assay showed that... Figure 14 The results showed that both G17 and G15 significantly enhanced TCR / NFAT signaling by blocking TIGIT / PVR and PVRIG / PVRL2 interactions. Importantly, the potency and efficacy of the BsAbs were higher than those of the individual reference mAbs WBP364-BMK1 and WBPT117-BMK1, as well as their combinations. A summary of antibody activities reversing TCR / NFAT inhibition is shown in Table 4. Fold change was defined as the ratio of luciferase in the sample to luciferase in the absence of the antibody.

[0400] 3.11 Human primary NK cell killing assay

[0401] HT1080 is a human fibrosarcoma cell line expressing human PVR, PVRL2, and PVRL3. HT1080 cells expressing human PVR and PVRL2 were used as target cells, and primary human NK cells were used as effector cells. Primary human NK cells were isolated from human peripheral blood mononuclear cells (PBMCs) using human CD56 microbeads (Miltenyi Biotec, catalog number 130-050-401) via magnetic selection, according to the manufacturer's protocol. The isolated human NK cells (4 x 10⁻⁶ cells) were then serially diluted with antibody in the presence of the antibody. 4 (cells / well) and HT1080 cells (2x10) 4 Cells per well were co-cultured at 37°C and 5% CO2 for 8 hours. Target cell lysis was determined using an LDH-based cytotoxicity assay kit (Roche, catalog number 04744934001). Absorbance was read at 492 nm using an M5e microplate reader (Molecule Devices).

[0402] The results of NK kill test showed Figure 15The results showed that both G17 and G15 enhanced the cytotoxicity of primary NK cells in a dose-dependent manner, exhibiting higher potency than WBP364-BMK1 and WBPT117-BMK1 alone, as well as their combination. A summary of antibodies that enhance NK cell activity is shown in Table 4.

[0403] 3.12 Human primary T cell activation assay

[0404] Human primary CD8 cells were co-cultured with artificial APCs. + T cells are stimulated via binding to T cell receptors. Artificial APCs are engineered HT1080 cells constitutively expressing PVR and PVRL2, and transfected to express human TCR activators. Human CD8 microbeads were isolated from human PBMCs via magnetic selection using human CD8 microbeads (Miltenyi Biotec, catalog number 130-045-201) according to the manufacturer's protocol. + T cells. Isolate human CD8 cells. + T cells were co-cultured with human T activator CD3 / CD28 Dynabeads (Gibco, catalog number 11132D) for 7 days to induce exhaustion. After the magnetic beads were removed using a magnet, these exhausted T cells (1x10⁻¹²) were... 5 (cells / well) and HT1080 / TCR activator cells treated with mitomycin C (1x10 cells / well) 4 (Units / well) were co-cultured in the presence of serially diluted antibodies. After 5 days of incubation, the supernatant was collected for IFN-γ measurement by ELISA (capture antibody Thermo catalog number M700A, detection antibody Thermo catalog number M701B). Absorbance was measured using an M5e microplate reader (Molecule Devices).

[0405] The results of the T-cell activation assay showed that... Figure 16 The results showed that both G17 and G15 enhanced depleted human CD8+ in a dose-dependent manner. + T cell activation, which has higher potency than WBP364-BMK1 and WBPT117-BMK1 alone, or their combinations. A summary of antibodies that enhance T cell activity is shown in Table 4.

[0406] Table 4. Summary of antibody characterization

[0407]

[0408] ND: Undetermined; NA: Unavailable

[0409] 3.13 Antibody Development

[0410] (1) Thermal stability determination

[0411] Conformational stability is a crucial characteristic for successful antibody candidates. It can be assessed by measuring thermal stability using differential scanning fluorometry (DSF), a method sensitive to changes in protein folding. DSF measures the temperature of protein unfolding transition (Tm) based on changes in the fluorescence intensity of the environmentally sensitive dye SYPRO Orange.

[0412] DSF was performed in the appropriate preparation buffer using a Quant Studio 7 Flex real-time PCR instrument (Applied Biosystems). SYPRO orange dye (Invitrogen, catalog number S6651) was added to the antibody, and the mixture was transferred to a 96-well plate. The plate was then transferred to the Quant Studio 7 Flex instrument. ® A 7. Flex real-time PCR system was used, with a temperature range of 26°C to 95°C and a heating rate of 0.9°C / min. The first two temperatures for protein unfolding transitions were recorded as Tm1 and Tm2. QuantStudio was used. ® The real-time PCR software (version 1.3) calculates these two values ​​based on the melting curve.

[0413] The DSF results are shown in Table 5 and Figure 17. The DSF thermograms of BsAb show two transitions: Tm1, reflecting the dissolution of the CH2 domain, and Tm2, ​​reflecting the melting temperatures of CH3 and Fab. The Tm1 values ​​for G17 and G15 are 57.1°C and 55.6°C, respectively, while the Tm2 values ​​are 64.7°C and 63.6°C, respectively. These results indicate that both G17 and G15 exhibit good thermal stability.

[0414] Table 5. Tm values ​​of antibodies

[0415]

[0416] (2) Solubility test using DLS-kD

[0417] Dynamic light scattering (DLS) is widely used to assess protein aggregation and stability. The diffusion interaction parameter (kD) describes how protein concentration affects the diffusion rate of a protein as a result of intermolecular forces.

[0418] To determine the kD value of the antibodies, DLS was performed using a DynaPro plate reader III (Wyatt Technology). A series of antibody concentrations (20, 15, 10, 5, and 2.5 mg / mL) were prepared, and 7.5 μL of the sample dilution was added to 1536-well microplates. The plates were sealed with ClearSeal membranes (Hampton Research, catalog number HR4-521) and centrifuged at 3,000 rpm for 5 minutes to remove aggregates. Each sample was tested in duplicate. Plates were read using a DynaPro1 plate reader III (Wyatt Technology), and data were collected using DYNAMICS operating software (v7.8.1.3). To obtain the kD value, the diffusion coefficient was determined and plotted against sample concentration. The slope of the plotted line represents the kD value of the sample.

[0419] The DLS-kD results are shown in Table 6 and Figure 18. The kD values ​​for G17 and G15 are -7.29 and -7.24 mL / g, respectively. No aggregation or particles were observed during the experiment. The results indicate good solubility for both G17 and G15.

[0420] Table 6. Antibody Solubility

[0421]

[0422] (3) Hydrophobicity test by HIC-HPLC

[0423] Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) is a powerful analytical method that has been used to predict the relative hydrophobicity of therapeutic proteins. The retention time of a protein of interest reflects its overall hydrophobicity.

[0424] To predict the hydrophobicity of antibodies, an HPLC 1260 Infinity II system (Agilent Technologies) was used. TMRetention times were calculated using a TSKgel butyl-NPR column (Tosoh, catalog number 0042168). Each sample was diluted to 0.5 mg / mL in PBS and 20 μL was injected into the column, with a separation flow rate of 0.5 mL / min for 61 min. Run buffers were prepared by mixing buffer A (25 mM sodium phosphate, pH 7.0) and buffer D (25 mM sodium phosphate, 1.5 M (NH4)2SO4, pH 7.0). Separation was performed from 3 to 53 min using a run buffer gradient (from 0% to 100% of buffer D). UV absorbance was determined at 280 nm and 230 nm to determine peak retention. Retention times were calculated by integrating all peak areas from 20 min to 40 min using OpenLab CDSWorkstation software (v2.6.0.691).

[0425] The results of HIC-HPLC are shown in Table 7 and Figure 19. The retention times of G17 and G15 were 30.31 and 28.62 min, respectively, which reflects normal hydrophobicity.

[0426] Table 7. Hydrophobicity of Antibodies

[0427]

[0428] (4) Stress tests at 4°C and 40°C

[0429] To investigate the thermal stability of BsAb, T4U1.G15 and T4U1.G17 were incubated in formulation buffer (20 mM histidine, 200 mM arginine, 70 mM sucrose, and 0.01% S80, pH 7.0) at 4°C and 40°C for up to 28 days. Samples were collected on days 0, 3, 7, 14, and 28 and stored at -40°C until analysis. The physical appearance of the samples was analyzed by observation, the concentration was analyzed by UV absorbance at 280 nm, and the changes in high molecular weight (HMW), monomer (Mono), and low molecular weight (LMW) species in the solution were analyzed by SEC-HPLC.

[0430] The results are shown in Tables 8 and 9. Under 40°C heat stress, the percentage of LMW increased slightly for G17 and decreased slightly for Mono, and for G15, the percentage of HMW increased slightly and decreased slightly for Mono. There were no significant changes in protein concentration.

[0431] Table 8. Stability of T4U1.G17 under thermal stress

[0432]

[0433] Table 9. Stability of T4U1.G15 under thermal stress

[0434]

[0435] (5) Stress tests after multiple freeze-thaw cycles

[0436] To investigate the stability of BsAb after multiple freeze-thaw (F / T) cycles, T4U1.G15 and T4U1.G17, stored at -80°C, were thawed at ambient temperature, refrozen at -80°C, and thawed again. After a total of 5 F / T cycles, samples were collected to evaluate the physical appearance by observation, the concentration by UV absorbance at 280 nm, and the changes in the types of HMW, Mono, and LMW present in the solution by SEC-HPLC.

[0437] The results are shown in Tables 10 and 11. After 5 F / T cycles, there were no perceptible effects on the stability of G17 and G15, or on the assays (including appearance, concentration, and the relative proportions of HMW, Mono, and LWM species).

[0438] Table 10. Stability of T4U1.G17 under freeze-thaw stress

[0439]

[0440] Table 11. Stability of T4U1.G15 under freeze-thaw stress

[0441]

[0442] Example 4

[0443] In vivo characterization

[0444] 4.1 Exploratory 4-week pharmacokinetic study in cynomolgus monkeys

[0445] The aim of this study was to evaluate the pharmacokinetic profile of anti-TIGIT / PVRIG BsAb after repeated intravenous infusions in naïve male and female cynomolgus monkeys.

[0446] This study used eight healthy cynomolgus monkeys (4 males and 4 females). Basic information is shown in Table 12.

[0447] Table 12. Basic Information on Crab-Eating Mammals

[0448]

[0449] Bispecific antibodies W3XX104-T4U1.G15-2.uIgG1 or W3XX104-T4U1.G17-2.uIgG1 were administered intravenously at doses of 50 or 125 mg / kg in one male and one female cynomolgus monkey. The dosing regimen was once weekly for four weeks (administered via IV infusion on days 0, 7, 14, and 21). This was a non-terminal study, and no necropsy was planned. Animals were returned to a relaxation room for washing and relaxation. Study endpoints included daily observations, body weight, serum cytokine concentrations, and pharmacokinetics.

[0450] Animal body weight was recorded twice a week. Serum samples were collected at specified time points (days -7, -2, and 0 after the first dose; day 1, 2, 3, 5, and 7 (before administration); day 11 and 14 (before administration); day 18 and 21 (before administration, and 1 and 8 hours after administration); and day 22, 23, 24, 26, and 28) for pharmacokinetic analysis. After centrifugation, serum was collected and stored at -80°C until transport for analysis. The concentration of testable BsAb in monkey serum was analyzed by ELISA. In summary, goat anti-human IgG (SouthernBiotech, catalog number 2049-01) was used as the capture reagent, and biotinylated goat anti-human IgG (SouthernBiotech, catalog number 2049-08) was used as the detection reagent. HRP-labeled streptavidin (Thermo, catalog number SNN1004) and TMB substrate were used for colorimetric development, and the reaction was terminated with 2M HCl. Absorbance was read at 450 nm and 540 nm using an M5e microplate reader (Molecule Devices). Serum concentrations of the assayable BsAb were determined by four-parameter fitting of a standard curve. Non-compartmental pharmacokinetic analysis of the concentrations was performed using Phoenix WinNonlin software (version 8.1, Pharsight). Linear / logarithmic trapezoidal rules were applied in the calculation of PK parameters.

[0451] Intravenous infusion of W3XX104-T4U1.G17-2.uIgG1 and W3XX104-T4U1.G15-2.uIgG1 was well tolerated in cynomolgus monkeys (at doses of 50 and 125 mg / kg, once weekly for 4 weeks), and all animals survived until the end of the study.

[0452] The bispecific antibodies T4U1.G17 and T4U1.G15 did not cause any significant changes in body weight during the study period. Figure 20 ).

[0453] Exposure in all animals was confirmed by measuring serum PK levels. PK profile is as follows: Figure 21-24 As shown, the PK parameters are summarized in Table 13. For T4U1.G17, the mean Cmax values ​​for 50 mg / kg and 125 mg / kg were 1890 µg / mL and 4980 µg / mL, respectively, while the mean AUC values ​​for 50 mg / kg and 125 mg / kg were... 0-t The values ​​were 155,645 h*µg / mL and 539,834 h*µg / mL, respectively. For T4U1.G15, the mean Cmax values ​​at 50 mg / kg and 125 mg / kg were 1335 µg / mL and 3305 µg / mL, respectively, while the mean AUC values ​​at 50 mg / kg and 125 mg / kg were... 0-t The values ​​were 266,105 h*µg / mL and 945,971 h*µg / mL, respectively. After repeated weekly administration to monkeys for 4 weeks, the Cmax values ​​of both T4U1.G17 and T4U1.G15 increased in a dose-dependent manner, and the AUC values ​​were... 0-t The values ​​increased in a manner greater than the dose-proportional ratio. Based on serum concentrations at four doses (days 1 to 28), the mean half-lives of T4U1.G17 were 54.5 and 56.3 hours for the 50 mg / kg and 125 mg / kg groups, respectively, while those of T4U1.G15 were 63.5 and 72.5 hours, respectively. Based on serum concentrations at one dose (days 1 to 7), the mean half-lives of T4U1.G17 were 55.1 and 73.9 hours for the 50 mg / kg and 125 mg / kg groups, respectively, while those of T4U1.G15 were 90.8 and 94.5 hours, respectively. No sex differences were observed within the study doses of 50 and 125 mg / kg.

[0454] Table 13 Summary of PK parameters

[0455]

[0456]

[0457] 4.2 In vivo efficacy of anti-TIGIT / PVRIG BsAb in CT26 colon cancer cell model

[0458] This study was conducted to evaluate the in vivo efficacy of the test substance in a syngeneic CT26 colon cancer cell model in human TIGIT and PVRIG transgenic BALB / c mice (BALB / c-hTIGIT / hPVRIG mice). CT26 cells (1x10⁻¹²) were used. 6100 μL / cell / mouse was subcutaneously injected into 6-8 week old BALB / c-hTIGIT / hPVRIG mice (provided by GemPharmatech). The tumor volume was measured at a mean tumor volume of 65.85 mm. 3 Forty tumor-bearing mice were randomly divided into 5 groups (n=8 / group) according to tumor volume. Mice in group G1 were administered PBS. Mice in groups G2-G4 were administered W3XX104-T4U1.G15-2.uIgG1 at 1.3 mpk, 4 mpk, and 13.2 mpk. Mice in group G5 were administered WBPT117-BMK1 (COM701) at 10 mpk and anti-TIGITmAb WBP364-BMK1 (tisrelimumab) at 10 mpk. COM701 was generated according to the sequence in patent US 2019 / 0382477 A1 (Table 1, clone ID: CHA.7.518.1). Tirelimumab was generated according to the sequence in INN PROP. LIST 117.

[0459] All mice were administered five doses intraperitoneally (ip) twice a week (BIW). Tumor volume and body weight were measured twice a week (D0, D4, D7, D11, D14, D17). Tumor volume was expressed in mm using the following formula. 3 This means: TV = 0.5 axb 2 Where a and b are the long and short diameters of the tumor, respectively. The tumor growth inhibition rate (TGI) based on tumor volume is measured. TV ) and tumor growth inhibition rate based on tumor weight (TGI) TW The in vivo efficacy of the test product was evaluated. Data are presented as mean ± standard error (mean ± SEM). Independent samples t-tests were performed to compare between two groups. P < 0.05 was considered statistically significant. GraphPad Prism 9 was used for data visualization.

[0460] Table 14. Experimental Design

[0461]

[0462] Weight changes in different groups are shown Figure 28 No significant weight loss was observed in either group. Tumor volume changes in mice across different groups were observed... Figure 25 , Figure 26 And in Table 15. Changes in tumor weight in mice across different groups are shown in... Figure 27 The statistical analysis of tumor growth inhibition and P-values ​​in different groups is shown in Table 16.

[0463] Table 15. TGI TV change

[0464]

[0465] Statistical analysis based on tumor volume at the endpoint (D17) showed that W3XX104-T4U1.G15-2.uIgG1 exhibited dose-dependent tumor growth inhibition at dosing levels of 1.3, 4, and 13.2 mg / kg, with TGI... TV The tumor suppression rates were 62.08%, 76.75%, and 86.69% respectively (W3XX104-T4U1.G15-2.uIgG1 treatment group vs. mediator control group, P<0.05). Furthermore, the tumor suppression in the W3XX104-T4U1.G15-2.uIgG1 monotherapy group (13.2 mg / kg) was comparable to that in the COM701 and tireliumab combination group (10+10 mg / kg) (TGI: 87.30% vs. 86.69%, P>0.05).

[0466] Table 16. Statistical analysis of tumor weight

[0467]

[0468] Note: Data are presented as mean ± SEM; statistics were performed using an independent samples t-test, *: P<0.05; **: P<0.01; ***: P<0.001.

[0469] Statistical analysis based on tumor weight at the endpoint (day 17) showed that W3XX104-T4U1.G15-2.uIgG1 exhibited dose-dependent tumor growth inhibition at dosing levels of 1.3, 4, and 13.2 mg / kg, with TGI... TW The tumor suppression rates were 65.29%, 77.44%, and 89.04% respectively (W3XX104-T4U1.G15-2.uIgG1 treatment group vs. mediator control group, P<0.05). Furthermore, the tumor suppression in the W3XX104-T4U1.G15-2.uIgG1 monotherapy group (13.2 mg / kg) was comparable to that in the COM701 and tireliumab combination group (10+10 mg / kg) (TGI). TW (89.04% and 88.05%, P>0.05).

[0470] In summary, the study demonstrates that, compared to the mediator control, W3XX104-T4U1.G15-2.uIgG1 dose-dependently (1.3 mpk, 4 mpk, and 13.2 mpk) inhibited tumor volume (P<0.01) and tumor weight (P<0.001). Furthermore, compared to combination therapy with COM701 and tislelizumab (G5, COM701 + tislelizumab, 10 mpk + 10 mpk), monotherapy with W3XX104-T4U1.G15-2.uIgG1 (G4, 13.2 mpk W3XX104-T4U1.G15-2.uIgG1, equivalent in molecular number to 10 mpk COM701 or tislelizumab) resulted in comparable tumor suppression.

[0471] Those skilled in the art will further understand that this disclosure may be embodied in other specific forms without departing from the spirit or central attributes of this disclosure. Since the foregoing description of this disclosure only discloses exemplary embodiments thereof, it should be understood that other variations should be considered within the scope of this disclosure. Therefore, the invention is not limited to the specific embodiments already described in detail herein. Rather, reference should be made to the appended claims, which indicate the scope and content of the invention.

[0472] References

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Claims

1. A polypeptide complex comprising a TIGIT binding moiety and a PVRIG binding moiety, wherein: the TIGIT binding moiety comprises a heavy chain variable region (VH) comprising a heavy chain CDR (HCDR) 1, a HCDR2, and a HCDR3, and a light chain variable region (VL) comprising a light chain CDR (LCDR) 1, a LCDR2, and a LCDR3, wherein: the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having no more than 1, 2, or 3 substitutions of amino acids compared to SEQ ID NO: 1, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having no more than 1, 2, or 3 substitutions of amino acids compared to SEQ ID NO: 2, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having no more than 1, 2, or 3 substitutions of amino acids compared to SEQ ID NO: 3, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having no more than 1, 2, or 3 substitutions of amino acids compared to SEQ ID NO: 4, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having no more than 1, 2, or 3 substitutions of amino acids compared to SEQ ID NO: 5, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having no more than 1, 2, or 3 substitutions of amino acids compared to SEQ ID NO: 6; wherein the substitutions are conservative substitutions.

2. The polypeptide complex of claim 1, wherein the PVRIG binding moiety comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3, and a VL comprising a LCDR1, a LCDR2, and a LCDR3, wherein: the HCDR1 comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having no more than 1, 2, or 3 substitutions compared to SEQ ID NO: 7, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having no more than 1, 2, or 3 substitutions compared to SEQ ID NO: 8, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having no more than 1, 2, or 3 substitutions compared to SEQ ID NO: 9, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having no more than 1, 2, or 3 substitutions compared to SEQ ID NO: 10, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having no more than 1, 2, or 3 substitutions compared to SEQ ID NO: 11, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having no more than 1, 2, or 3 substitutions compared to SEQ ID NO: 12, wherein the substitutions are conservative substitutions.

3. The polypeptide complex of claim 1 or 2, wherein the TIGIT-binding moiety and the PVRIG-binding moiety are in the form of any one of a Fab and a scFv.

4. The polypeptide complex of claim 1 or 2, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the TIGIT-binding moiety comprise the amino acid sequences of SEQ ID NOs: 1-6, respectively; and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the PVRIG-binding moiety comprise the amino acid sequences of SEQ ID NOs: 7-12, respectively.

5. The polypeptide complex of any one of claims 1-4, wherein the VH of the TIGIT-binding moiety comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 13, and / or the VL of the TIGIT-binding moiety comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO:

14.

6. The polypeptide complex of any one of claims 1-5, wherein the VH of the PVRIG-binding moiety comprises the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 15, and / or the VL of the PVRIG-binding moiety comprises the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO:

16.

7. The polypeptide complex of any one of the preceding claims, wherein: the TIGIT-binding moiety is in the form of a Fab, and the PVRIG-binding moiety is in the form of a scFv; the TIGIT-binding moiety is in the form of a scFv, and the PVRIG-binding moiety is in the form of a Fab; or both the TIGIT-binding moiety and the PVRIG-binding moiety are in the form of a Fab.

8. The polypeptide complex of any one of the preceding claims, wherein the polypeptide complex further comprises an immunoglobulin constant region, such as an IgG constant region, such as a human IgGl, IgG4, IgG2, IgG3 Fc region or a variant thereof.

9. The polypeptide complex of claim 8, wherein the human IgG Fc region is a human IgGl Fc region or a variant thereof.

10. The polypeptide complex of claim 9, wherein the variant comprises one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or prolong its half-life.

11. The polypeptide complex of any one of claims 8-10, wherein the polypeptide complex comprises: (a) the TIGIT-binding moiety operably linked to the Fc region, and the PVRIG-binding moiety operably linked to the Fc region; (b) the TIGIT-binding moiety operably linked to the PVRIG-binding moiety, and the PVRIG-binding moiety operably linked to the Fc region; or (c) the PVRIG-binding moiety operably linked to the TIGIT-binding moiety, and the TIGIT-binding moiety operably linked to the Fc region.

12. The polypeptide complex of any one of the preceding claims, comprising one, two, or more TIGIT-binding moieties, and one, two, or more PVRIG-binding moieties.

13. The polypeptide complex of claim 12, wherein the TIGIT-binding moieties are the same or different, and / or the PVRIG-binding moieties are the same or different.

14. The polypeptide complex of any one of the preceding claims, comprising two heavy chains and two light chains, wherein the TIGIT-binding moiety is in the form of a Fab, and the PVRIG-binding moiety is in the form of a scFv, and wherein from N- to C-terminus: each of the first and second heavy chains comprises domains operably linked in the form of: VH1-CH1-Fc-scFv or scFv-VH1-CH1-Fc, each of the first and second light chains comprises domains operably linked in the form of: VL1-CL, wherein the VH1-CH1 and the VL1-CL are from the TIGIT-binding moiety, and the scFv is from the PVRIG-binding moiety. ​ 15. The polypeptide complex of any one of claims 1-13, comprising two heavy chains and two light chains, wherein the TIGIT binding moiety is in the form of a scFv and the PVRIG binding moiety is in the form of a Fab, and wherein from N- to C-terminus: each of the first and second heavy chains comprises domains operably linked in the form of: scFv-VH2-CH1-Fc or VH2-CH1-Fc-scFv, each of the first and second light chains comprises domains operably linked in the form of: VL2-CL, wherein the scFv is from the TIGIT binding moiety and the VH2-CH1 and VL2-CL are from the PVRIG binding moiety.

16. The polypeptide complex of any one of claims 1-13, comprising two heavy chains and four light chains, wherein the TIGIT binding moiety and the PVRIG binding moiety are in the form of a Fab, and wherein from N- to C-terminus: each of the first and second heavy chains comprises domains operably linked in the form of: VH1-CH1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-CH1, VH1-CH1-VH2-CH1-Fc or VH2-CH1-VH1-CH1-Fc, each of the first and second light chains comprises domains operably linked in the form of: VL1-CL, each of the third and fourth light chains comprises domains operably linked in the form of: VL2-CL, wherein the VH1-CH1 and VL1-CL are from the TIGIT binding moiety and the VH2-CH1 and VL2-CL are from the PVRIG binding moiety.

17. The polypeptide complex of any one of claims 1-13, comprising two heavy chains and four light chains, wherein the TIGIT binding moiety and the PVRIG binding moiety are in the form of a Fab, and wherein from N- to C-terminus: each of the first and second heavy chains comprises domains operably linked in the form of: VH1-C1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-C1, VH1-C1-VH2-CH1-Fc or VH2-CH1-VH1-C1-Fc, each of the first and second light chains comprises domains operably linked in the form of: VL1-C2, each of the third and fourth light chains comprises domains operably linked in the form of: VL2-CL, wherein the VH1-C1 and VL1-C2 are from the TIGIT binding moiety and the VH2-CH1 and VL2-CL are from the PVRIG binding moiety.

18. The polypeptide complex of any one of claims 1-13, comprising two heavy chains and four light chains, wherein the TIGIT binding moiety and the PVRIG binding moiety are in the form of a Fab, and wherein from N- to C-terminus: each of the first and second heavy chains comprises domains operably linked in the following format: VH1-CH1-Fc-VH2-C1, VH2-C1-Fc-VH1-CH1, VH1-CH1-VH2-C1-Fc, or VH2-C1-VH1-CH1-Fc, each of the first and second light chains comprises domains operably linked in the following format: VL1-CL, each of the third and fourth light chains comprises domains operably linked in the following format: VL2-C2, wherein said VH1-CH1 and said VL1-CL are from said TIGIT-binding moiety, and said VH2-C1 and said VL2-C2 are from said PVRIG-binding moiety.

19. The polypeptide complex according to any one of claims 7-18, wherein the scFv comprises a VH region operably linked to a VL region, and the VH region is N-terminal to the VL region or the VL region is N-terminal to the VH region.

20. The polypeptide complex according to any one of claims 11-19, wherein operable linkage is via a direct linkage or via a peptide linker.

21. The polypeptide complex according to claim 20, wherein the peptide linker is a hinge region or a GS linker, such as (GS)n, (GGS)n, (GGGS)n, (GGGGS)n, (GGSG)n, (GGGSS)n, wherein n is an integer from 1-9.

22. The polypeptide complex according to any one of claims 14-15, comprising: (i) a first and second heavy chain comprising SEQ ID NO: 17, and a first and second light chain comprising SEQ ID NO: 18; or (ii) a first and second heavy chain comprising SEQ ID NO: 19, and a first and second light chain comprising SEQ ID NO:

20.

23. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the heavy chain variable region and / or the light chain variable region of the polypeptide complex according to any one of claims 1-22.

24. A vector comprising the isolated nucleic acid molecule according to claim 23.

25. A host cell comprising the vector according to claim 24 or the isolated nucleic acid molecule according to claim 23.

26. A pharmaceutical composition comprising the polypeptide complex according to any one of claims 1-22 and a pharmaceutically acceptable carrier.

27. A method for producing the polypeptide complex according to any one of claims 1-22, comprising the steps of: - culturing a host cell comprising an expression vector encoding the polypeptide complex under suitable conditions; and - harvesting the polypeptide complex from the cell culture.

28. A method for treating or preventing a cancer or an immune-related disorder in a subject, such as a human and a non-human mammal such as a mouse, comprising administering to the subject an effective amount of the polypeptide complex according to any one of claims 1-22 or the pharmaceutical composition according to claim 26.

29. The method of claim 28, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, renal clear cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, mesothelioma, myeloma, and sarcoma, for example the cancer is colon cancer.

30. The method of claim 28, wherein the immune-related disorder is a T cell dysfunction disorder or an infection.

31. The method of any one of claims 29-30, wherein the method further comprises administering to the subject an additional therapeutic agent.

32. The polypeptide complex of any one of claims 1-22 for use in the treatment or prevention of a cancer or a T cell dysfunction disorder or an infection.

33. Use of the polypeptide complex of any one of claims 1-22 in the manufacture of a medicament for the treatment or prevention of a cancer or a T cell dysfunction disorder or an infection.

34. The polypeptide complex for use according to claim 32 or the use according to claim 33, wherein the cancer is selected from colon cancer, lung cancer, breast cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, renal clear cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, neoplasm of the central nervous system, mesothelioma, myeloma, and sarcoma, for example the cancer is colon cancer.

35. A kit comprising a container comprising the polypeptide complex of any one of claims 1-22.

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