Anti-TIGITxPVRIG antibody and its use

Abstract

The present invention provides an anti-TIGIT / PVRIG antibody and polypeptide complex, an encoding nucleic acid molecule, and an expression vector and host cells used for the expression of the above antibody. Furthermore, the present invention provides a method for preventing and treating cancer and immunodeficiency by administering the above anti-TIGIT / PVRIG antibody.

Description

[Technical Field]

[0001] <Cross reference> This application claims priority to international patent application PCT / CN2023 / 098602, filed on 6 June 2023, the entire contents of said application are incorporated herein by reference.

[0002] <Sequence Listing> This application includes a sequence listing, the entire contents of which are incorporated herein by reference.

[0003] <Technical field> This application relates to antibodies in general. More specifically, it relates to antibodies and polypeptide conjugates targeting TIGIT and PVRIG, methods for preparing them, and the use of these antibodies. [Background technology]

[0004] In the tumor microenvironment, sustained antigen stimulation can lead to T cell exhaustion (a state of T cell dysfunction), and co-inhibitory receptors such as PD-1, LAG-3, TIM3, and TIGIT may be overexpressed. Currently, numerous strategies are being investigated to reactivate exhausted T cells using small molecules or therapeutic antibodies, either alone or in combination.

[0005] TIGIT (T cell immune receptor with Ig and ITIM domains), also known as Vstm3 and WUCAM, is a co-inhibitory receptor expressed on NK and CD8+ T cells, as well as CD4+ T cell subsets such as immunosuppressive regulatory T cells (Tregs) [1-4]. Four ligands for TIGIT are known: poliovirus receptor (PVR), PVRL2, PVRL3, and PVRL4, all of which are overexpressed in tumors and antigen-presenting cells, causing immunosuppression [3, 5-8]. These ligands also bind to the co-stimulatory molecule CD226 and the co-inhibitory molecules PVRIG and CD96 (the latter sometimes considered a co-stimulatory molecule). TIGIT antagonist antibodies prevent TIGIT from binding to its ligand, blocking its inhibitory signal and inducing a potent anti-tumor immune response by making CD226-mediated activation signals dominant [9-11]. TIGIT, like other exhaustion markers such as PD-1, LAG3, and TIM3 [15, 16], is elevated in cancer and inflammatory diseases and has been identified as an exhaustion marker [12-14]. Furthermore, TIGIT has been identified as an important inhibitory receptor for stem cell-like memory T cells, a new population of T cells that may be a preferred target for anti-PD-(L)1 efficacy [17, 18]. TIGIT may be a promising therapeutic target in tumor immunotherapy, either as a monotherapy or in combination with other immunomodulators.

[0006] On the one hand, the poliovirus receptor-related Ig domain-containing protein (PVRIG, also known as CD112R) is one of the co-inhibitory immune checkpoint proteins. PVRIG plays an important role in the recovery of T cell exhaustion and the increase in NK cell activation [19-22]. PVRIG belongs to the nectin and nectin-like families, and these family members also include TIGIT, DNAM-1 (CD226), and CD96. PVRIG is expressed in NK cells and T cells, and its expression further increases in T cells when activated [19, 20]. The interaction between PVRIG and PVRL2 (CD112), the ligand of PVRIG expressed in APCs and multiple tumor cells, suppresses the activation of T cells and NK cells [21, 22]. PVRL2 is also a ligand of CD226, and CD226 activates human T cells and human NK cells through interaction with the ligand [23-25]. Although PVRIG and TIGIT have many similarities, distinct differences indicating that these pathways are not redundant have been reported by scientists [20, 22]. Targeting these pathways may synergistically enhance the anti-tumor response.

[0007] Blocking immune checkpoints (anti-CTLA-4 antibody, anti-PD-1 antibody, and anti-PD-L1 antibody) can bring the possibility of durable remission to patients with 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 being so widely applicable, the majority of cancer patients (well over 80%) will ultimately show refractoriness or resistance to CTLA-4, PD-1 / PD-L1 immune checkpoint therapy.

[0008] Therefore, there is a need for novel antibodies that can effectively target immune checkpoint molecules and restore T cell function for cancer treatment. Summary of the Invention Means for Solving the Problems

[0009] These and other objects are provided by the present disclosure, which in a broad sense relates to compounds, methods, compositions, and products that provide antibodies with improved efficacy. The advantages provided by the present disclosure are widely applicable in the fields of antibody pharmaceuticals and diagnostics and can be used in combination with other antibodies that react with various targets.

[0010] In some embodiments, the present disclosure provides a polypeptide complex, such as a bispecific polypeptide complex, that can bind to the proteins or antigens of TIGIT and PVRIG. The proteins of TIGIT and PVRIG may be particularly derived from, for example, humans, cynomolgus monkeys, or mice. In some embodiments, the polypeptide complex has interspecies reactivity and can, for example, bind to human TIGIT, cynomolgus monkey TIGIT, and mouse TIGIT. In some embodiments, the polypeptide complex can bind to human PVRIG and cynomolgus monkey PVRIG.

[0011] In some embodiments, the polypeptide complex includes a TIGIT-binding portion and a PVRIG-binding portion. The TIGIT-binding portion includes heavy chain CDRs (HCDRs) 1, 2, and 3 and light chain CDRs (LCDRs) 1, 2, and 3, and the HCDRs 1, 2, and 3 each include, consist essentially of, or consist of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the LCDRs 1, 2, and 3 each include, consist essentially of, or consist of the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, and The above PVRIG binding portion includes HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, where HCDR1, HCDR2, and HCDR3 each contain, essentially consist of, or consist of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, and LCDR1, LCDR2, and LCDR3 each contain, essentially consist of, or consist of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0012] In some embodiments, the polypeptide complex includes a TIGIT binding site and a PVRIG binding site. The above TIGIT connection portion is It comprises a heavy chain CDR (HCDR)1 containing the amino acid sequence of SEQ ID NO: 1, HCDR2 containing the amino acid sequence of SEQ ID NO: 2, HCDR3 containing the amino acid sequence of SEQ ID NO: 3, a light chain CDR (LCDR)1 containing the amino acid sequence of SEQ ID NO: 4, LCDR2 containing the amino acid sequence of SEQ ID NO: 5, and LCDR3 containing the amino acid sequence of SEQ ID NO: 6. The above PVRIG coupling portion is It includes 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, 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.

[0013] In some embodiments, the TIGIT coupling portion is (A) (i) The amino acid sequence shown in Sequence ID No. 13, (ii) an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 13, or (iii) Amino acid sequences having one or more additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 13 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid). The heavy chain variable region (VH) including, (B) (i) The amino acid sequence shown in Sequence ID No. 14, (ii) an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 14, or (iii) Amino acid sequences having one or more additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 14 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid). Light chain variable region (VL) including Includes.

[0014] In some embodiments, the PVRIG coupling portion is (A) (i) The amino acid sequence shown in Sequence ID No. 15, (ii) an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 15, or (iii) Amino acid sequences having one or more additions, deletions and / or substitutions of amino acids compared to SEQ ID NO: 15 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid) The heavy chain variable region (VH) including, (B) (i) The amino acid sequence shown in Sequence ID No. 16, (ii) an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 16, or (iii) Amino acid sequences having one or more additions, deletions and / or substitutions of amino acids (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1) compared to SEQ ID NO: 16 Light chain variable region (VL) including Includes.

[0015] In some embodiments, the TIGIT coupling portion includes HCDR1, HCDR2, and HCDR3 in the VH region shown in SEQ ID NO: 13, and LCDR1, LCDR2, and LCDR3 in the VL region shown in SEQ ID NO: 14. In some embodiments, the PVRIG coupling portion includes HCDR1, HCDR2, and HCDR3 in the VH region shown in SEQ ID NO: 15, and LCDR1, LCDR2, and LCDR3 in the VL region shown in SEQ ID NO: 16.

[0016] In some embodiments, the TIGIT binding portion includes a VH region containing the amino acid sequence of SEQ ID NO: 13 and a VL region containing the amino acid sequence of SEQ ID NO: 14. In some embodiments, the PVRIG binding portion includes a VH region containing the amino acid sequence of SEQ ID NO: 15 and a VL region containing the amino acid sequence of SEQ ID NO: 16.

[0017] In some embodiments, the TIGIT coupling portion is in Fab or scFv format. In some embodiments, the PVRIG coupling portion is in Fab or scFv format. In some embodiments, the TIGIT coupling portion is in Fab format and the PVRIG coupling portion is in scFv format. In some other embodiments, the TIGIT coupling portion is in scFv format and the PVRIG coupling portion is in Fab format.

[0018] In some embodiments, the polypeptide complex further comprises a human IgG constant region, such as a human IgG1, IgG4, IgG2, or IgG3 constant region. The human IgG constant region may be a natural IgG Fc region or a variant thereof. In some embodiments, the polypeptide complex comprises a natural human IgG1 Fc region.

[0019] In some embodiments, the variant of the Fc region includes one or more substitutions to modulate receptor binding or effector function, promote dimerization, prevent glycosylation, and / or extend the antibody half-life.

[0020] In some embodiments, the polypeptide complex comprises one, two, or more TIGIT binding sites and one, two, or more PVRIG binding sites. The TIGIT binding sites may be the same or different, and / or the PVRIG binding sites may be the same or different. The polypeptide complex may be a homodimer or a heterodimer.

[0021] In some embodiments, the polypeptide complex comprises two heavy chains and two light chains, the TIGIT binding portion is in Fab format, the PVRIG binding portion is in scFv format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain operably linked domains such as VH1-CH1-Fc-scFv or scFv-VH1-CH1-Fc, The first light chain and the second light chain each contain domains that are operablely linked, such as VL1-CL. The above VH1-CH1 and VL1-CL are derived from the above TIGIT binding site, and the above scFv is derived from the above PVRIG binding site. VH1 and VL1 refer to the first VH and first VL in the polypeptide complex, and these constitute the TIGIT binding site. VH2 and VL2 refer to the second VH and second VL, and these constitute the PVRIG binding site.

[0022] In some embodiments, the polypeptide complex comprises two heavy chains and two light chains, the TIGIT binding portion is in scFv format, the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain operably linked domains such as scFv-VH2-CH1-Fc, The first light chain and the second light chain each contain domains that are operablely linked, such as VL2-CL. The above scFv is derived from the above TIGIT binding site, and the above VH2-CH1 and VL2-CL are derived from the above PVRIG binding site.

[0023] In some embodiments, the polypeptide complex comprises two heavy chains and four light chains, and the TIGIT binding and PVRIG binding portions are in Fab format, from the N-terminus to the C-terminus. The first heavy chain and the second heavy chain each contain operably linked domains such as VH1-CH1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-CH1, VH1-CH1-VH2-CH1-Fc, or VH2-CH1-VH1-CH1-Fc. The first light chain and the second light chain each contain domains that are operablely linked, such as VL1-CL. The third and fourth light chains each contain domains that are operablely linked, such as VL2-CL. The above VH1-CH1 and VL1-CL are derived from the above TIGIT binding site, and the above VH2-CH1 and VL2-CL are derived from the above PVRIG binding site.

[0024] In some embodiments, the polypeptide complex comprises two heavy chains and four light chains, the TIGIT binding portion and the PVRIG binding portion are in Fab format, and the TIGIT binding portion is a chimeric Fab comprising VH1 operably linked to a first T cell receptor (TCR) constant region (C1) and VL1 operably linked to a second TCR constant region (C2), and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain operably linked domains such as VH1-C1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-C1, VH1-C1-VH2-CH1-Fc, or VH2-CH1-VH1-C1-Fc. The first light chain and the second light chain each include domains that are operably linked, such as VL1-C2. The third and fourth light chains each contain domains that are operablely linked, such as VL2-CL. The above VH1-C1 and VL1-C2 are derived from the above TIGIT binding site, and the above VH2-CH1 and VL2-CL are derived from the above PVRIG binding site. C1 and C2 refer to a pair of TCR constant regions that can bind to each other to form a dimer, or engineered variants thereof that include one or more non-natural disulfide bonds.

[0025] In some embodiments, the polypeptide complex comprises two heavy chains and four light chains, the TIGIT binding portion and the PVRIG binding portion are in Fab format, and the PVRIG binding portion is a chimeric Fab comprising VH2 operably linked to C1 and VL2 operably linked to C2, from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain operably linked domains such as VH1-CH1-Fc-VH2-C1, VH2-C1-Fc-VH1-CH1, VH1-CH1-VH2-C1-Fc, or VH2-C1-VH1-CH1-Fc. The first light chain and the second light chain each contain domains that are operablely linked, such as VL1-CL. The third and fourth light chains each contain domains that are operably linked, such as VL2-C2. The above VH1-CH1 and VL1-CL are derived from the above TIGIT binding site, and the above VH2-C1 and VL2-C2 are derived from the above PVRIG binding site.

[0026] In some embodiments, the scFv includes a VH region operably linked to a VL region directly or via a linker, wherein the VH region is located at the N-terminus of the VL region, or the VL region is located at the N-terminus of the VH region.

[0027] In some embodiments, the above portion is operably linked to the Fc region directly or via a linker. The linker may be a peptide linker such as a hinge region (hereinafter referred to as "hinge") of all or part of the immunoglobulin, or a conventionally used artificial linker such as a GS linker. The GS linker used herein may 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 of the above disclosure is operably linked to the Fc region via a hinge region.

[0028] In some embodiments, the polypeptide complex is (i) A first heavy chain and a second heavy chain containing SEQ ID NO: 17, and a first light chain and a second light chain containing SEQ ID NO: 18, or (ii) A first heavy chain and a second heavy chain containing sequence number 19, and a first light chain and a second light chain containing sequence number 20.

[0029] In some embodiments, the Disclosure provides isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of polypeptide complexes disclosed herein.

[0030] In some embodiments, the present disclosure provides an expression vector comprising nucleic acid molecules disclosed herein.

[0031] In some embodiments, the present disclosure provides host cells comprising the expression vectors disclosed herein.

[0032] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a polypeptide complex disclosed herein and a pharmaceutically acceptable carrier.

[0033] In some embodiments, the present disclosure provides a method for preparing the polypeptide complex, comprising expressing the polypeptide complex in a host cell containing an expression vector encoding the polypeptide complex and isolating the polypeptide complex from the culture supernatant.

[0034] In some embodiments, the present disclosure provides a method for modulating a TIGIT / PVRIG-related immune response in a subject, comprising administering a polypeptide complex disclosed herein to the subject.

[0035] In some embodiments, the present disclosure provides a method for inhibiting the proliferation of tumor cells in a subject, comprising administering to the subject an effective amount of a polypeptide complex or pharmaceutical composition disclosed herein, either alone or in combination with another anticancer agent, such as an anti-PD-L1 antibody.

[0036] In some embodiments, the Disclosure provides a method for treating or preventing cancer or an immunodeficiency in a subject, comprising administering an effective amount of the polypeptide complex disclosed herein, either alone or in combination with another anticancer agent, to the subject.

[0037] The above anticancer agent may be a chemotherapeutic agent, a monoclonal antibody, an antibody-drug conjugate, etc. In some embodiments, the above anticancer agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0038] The cancers listed above can be selected from colon cancer, lung cancer (NSCLC, etc.), 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, stomach cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system tumors, mesothelioma, leukemias such as chronic lymphocytic leukemia, lymphomas such as diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma, myeloma, soft tissue cancer, and sarcoma. The above-mentioned immune deficiency may be T-cell dysfunction or infection.

[0039] In some embodiments, the polypeptide complexes disclosed herein are administered in combination with an anti-PD-L1 antibody.

[0040] In some embodiments, the present disclosure provides combinations of polypeptide conjugates disclosed herein with anti-PD-L1 antibodies such as atezolizumab.

[0041] In some embodiments, the Disclosure provides the use of the polypeptide conjugates disclosed herein, alone or in combination with other anticancer agents, in the manufacture of pharmaceuticals for treating or preventing diseases such as cancer and immunodeficiencies. In some embodiments, the anticancer agent is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0042] In some aspects, the disclosure provides the use of the polypeptide conjugates disclosed herein in the manufacture of diagnostic agents for diagnosing diseases associated with TIGIT / PVRIG overexpression.

[0043] In some embodiments, this disclosure provides polypeptide conjugates disclosed herein for use in the treatment or prevention of cancer and immunodeficiency. In some embodiments, the antibodies disclosed herein are used in combination with PD-1 / PD-L1 antagonists, such as PD-L1 antibodies.

[0044] In some embodiments, the Disclosure provides a kit or apparatus comprising one or more polypeptide complexes disclosed herein in one or more containers.

[0045] As the above is a summary, it naturally includes simplifications, generalizations, and omissions of details, and therefore, those skilled in the art will understand that this summary is merely illustrative and not intended to be limiting. This summary is not intended to identify the main or essential features of the subject matter of the claims, nor is it intended to be used as an aid in determining the scope of the subject matter of the claims. [Brief explanation of the drawing]

[0046] [Figure 1] Figure 1 shows schematic diagrams of the structures of W3XX104-T4U1.G15-2.uIgG1 and W3XX104-T4U1.G17-2.uIgG1. [Figure 2] Figure 2 shows the binding of antibodies to human TIGIT as determined by FACS. [Figure 3] Figure 3 shows the binding of antibodies to human PVRIG as determined by FACS. [Figure 4] Figure 4 shows the binding of antibodies to cynomolgus monkey TIGIT as determined by FACS. [Figure 5] Figure 5 shows the binding of antibodies to cynomolgus monkey PVRIG as determined by FACS. [Figure 6] Figure 6 shows the binding of antibodies to mouse TIGIT as determined by FACS. [Figure 7] Figure 7 shows the binding of antibodies to mouse PVRIG as determined by FACS. [Figure 8] Figure 8 shows the SPR sensorgram of antibodies that bind to human TIGIT. [Figure 9] Figure 9 shows the SPR sensorgram of an antibody that binds to human PVRIG. [Figure 10] Figure 10 shows the binding of antibodies to the paralog proteins TIGIT and PVRIG as determined by ELISA. [Figure 11] Figure 11 shows the results of double binding of antibodies to human TIGIT and PVRIG. [Figure 12] Figure 12 shows the results of blocking PVR binding to TIGIT by antibody, as determined by FACS. [Figure 13] Figure 13 shows the results of blocking the binding of PVRL2 to PVRIG by antibody, as determined by ELISA. [Figure 14]Figure 14 shows the antibody results in the Jurkat TIGIT / PVRIG / NFAT-luciferase reporter gene assay. [Figure 15] Figure 15 shows the effect of the antibody in the NK cell killing assay. [Figure 16] Figure 16 shows the effect of antibodies in a T cell activation assay. [Figure 17A] Figure 17A shows the melting curve of W3XX104-T4U1.G17-2.uIgG1. [Figure 17B] Figure 17B shows the melting curve of W3XX104-T4U1.G15-2.uIgG1. [Figure 18A] Figure 18A shows a plot of the diffusion coefficient against the concentration of W3XX104-T4U1.G17-2.uIgG1. [Figure 18B] Figure 18B shows a plot of the diffusion coefficient against the concentration of W3XX104-T4U1.G15-2.uIgG1. [Figure 19A] Figure 19A shows the retention of W3XX104-T4U1.G17-2.uIgG1 in HIC-HPLC. [Figure 19B] Figure 19B shows the retention of W3XX104-T4U1.G15-2.uIgG1 in HIC-HPLC. [Figure 20] Figure 20 shows the changes in body weight of monkeys during pharmacokinetic studies. [Figure 21] Figure 21 shows the PK profile of W3XX104-T4U1.G17-2.uIgG1 in pharmacokinetic studies in monkeys. [Figure 22] Figure 22 shows the PK profile of W3XX104-T4U1.G17-2.uIgG1 in pharmacokinetic studies in monkeys. [Figure 23] Figure 23 shows the PK profile of W3XX104-T4U1.G15-2.uIgG1 in pharmacokinetic studies in monkeys. [Figure 24] Figure 24 shows the PK profile of W3XX104-T4U1.G15-2.uIgG1 in pharmacokinetic studies in monkeys. [Figure 25] Figure 25 shows the mean change in tumor volume in the CT26 mouse model. [Figure 26] Figure 26 shows the individual tumor volume changes at treatment in different groups. [Figure 27] Figure 27 shows the tumor weights in different groups at the end of the study (day 17) in the CT26 mouse model. [Figure 28] Figure 28 shows the mean weight changes in different groups after administration in the CT26 mouse model. [Modes for carrying out the invention]

[0047] While the present invention can be embodied in many different forms, this specification discloses specific exemplary embodiments illustrating the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated. Furthermore, section headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0048] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified in the context, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used herein and in the appended claims, the singular forms "a," "an," and "the" shall include multiple references unless otherwise explicitly indicated in the context. Thus, for example, a reference to "a protein" includes multiple proteins, and a reference to "a cell" includes a mixture of cells. In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "comprising," as well as other forms such as "comprises" and "comprised," is not limited. Also, the scopes indicated herein and in the appended claims include both endpoints and all points between those endpoints.

[0049] In general, the nomenclature and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, as described herein are well known and commonly used in the art. The methods and techniques described herein are generally carried out in accordance with conventional methods well known in the art, unless otherwise specified, as described in the various general and more specific references cited and explained throughout this specification. For example, Abbas et al., Cellular and Molecular Immunology, 6 th See also 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, experimental procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well known and commonly used in the art.

[0050] definition To better understand this disclosure, the following definitions and explanations of relevant terms are provided.

[0051] As used herein, the term “antibody” or “Ab” is used in its broadest sense and encompasses any form of antibody exhibiting desired biological or binding activity. This includes, but is not limited to, humanized antibodies, fully human antibodies, chimeric antibodies, and single-domain antibodies. Bispecific polypeptide complexes disclosed herein also belong to the category of antibodies. A typical antibody usually comprises a heavy chain and a light chain. Heavy chains can be classified into 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 demonstrated herein, various modifications may be made to the constant regions, or they may 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)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, in the following order from the N-terminus to the C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The extent of the framework regions and CDRs can be precisely identified using methods known in the art, for example, by definitions such as Kabat, Chothia, AbM, EU, and / or Contact, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877, Chothia, C. et al. 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 USA.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. Correspondence or alignment between each numbering by different definitions 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 be different antibody isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0052] 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 an intact native antibody structure. Examples of antigen-binding moieties include, but are not limited to, variable domains, variable regions, diabodies, 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 diabodies (dsdiabodies), multispecific antibodies, camelized single-domain antibodies, single variable domains (i.e., VHH), nanobodies, domain antibodies, and bivalent domain antibodies. An antigen-binding moiety can bind to the same antigen to which the parent antibody binds. In certain embodiments, an antigen-binding moiety may contain one or more CDRs derived from a particular antibody grafted onto a framework region derived from a different antibody. Further detailed formats of the antigen-binding moiety are described 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.

[0053] In relation to an antibody, "Fab" refers to the portion of the antibody consisting of a single light chain (both variable and constant regions) attached by a disulfide bond to the variable region and first constant region of a single heavy chain. In certain embodiments, both the light and heavy chain constant regions of Fab may be replaced with the TCR constant regions Cα and Cβ or their engineered variants.

[0054] In relation to antibodies, "Fc" (short for crystallizable fragment) refers to a portion of the antibody containing the second and third constant regions (CH2 and CH3) of the first heavy chain, which are bound to the second and third constant regions of the second heavy chain via disulfide bonds. Fc is a specific dimerization domain. As used herein, the Fc region may also include 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 it does not typically function in antigen binding. The ability of an antibody to initiate and control effector functions via the Fc domain is an important component of its in vivo protective activity.

[0055] As used herein, the terms “monoclonal antibody” or “mAb” refer to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope.

[0056] As used herein, the term “humanized antibody” refers to an antibody in which a CDR sequence derived from the germline of another mammalian species, such as rat or mouse, is grafted onto a human framework sequence. Further framework region modifications can be performed within the human framework sequence. Humanized antibodies will also optionally include an immunoglobulin constant region (e.g., Fc), typically at least a portion of the human immunoglobulin constant region.

[0057] As used herein, the terms “human antibody” or “fully human antibody” are intended to include antibodies having variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody includes a constant region, that constant region is also derived from a human germline immunoglobulin sequence.

[0058] As used herein, the terms “TIGIT” or “T-cell immune receptor having Ig and ITIM domains” refer to any natural TIGIT derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. The term encompasses untreated full-length TIGIT, the extracellular domain of TIGIT, and all forms of TIGIT resulting from intracellular processing. The term also encompasses naturally occurring variants of TIGIT, such as splice variants and allele variants.

[0059] The terms “PVRIG” or “Poliovirus Receptor Related Immunoglobulin Domain Containing Protein” include known or wild-type PVRIG, as well as their variants, complexes, or fragments (particularly extracellular domain fragments). PVRIG is a transmembrane domain protein having a signal peptide, extracellular domain, transmembrane domain, and cytoplasmic domain. PVRIG is expressed on the cell surface of NK cells and T cells and shares some similarities with other known immune checkpoints. Identification and methods for demonstrating that PVRIG is a checkpoint receptor can be found in International Publication No. 2016 / 134333, incorporated herein by reference.

[0060] As used herein, the term "PD-1 / PD-L1 antagonist" includes PD-L1 antagonists (such as anti-PD-L1 antibodies) that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 or B7-1), and PD-1 antagonists (such as anti-PD-1 antibodies) that reduce, block, inhibit, suppress, or interfere with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners (such as PD-L1, PD-L2, etc.). In some embodiments, the PD-1 antagonist is nivolumab (MDX-1106) or pembrolizumab (formerly lambrolizumab (MK-3475)), MED1-0680, PDR001 (spartalizumab), REGN2810 (semiprimab), BGB-108, prorugolimab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, retifanlimab, spartalizumab, sasa Nlimab, Penplimab, CS1003, HLX10, SCT-I10A, SHR-1316, CS1001, Embafolimab, TQB2450, ZKAB001, LP-002, Zimbererimab, Valstilimab, Genolimuzumab, BI754091, Cetrelimab, YBL-006, BAT1306, HX008, CX-072, IMC-001, KL-A167, Budigalimab, AMG404, CX-188, J TX-4014, 609A, Sym021, LZM009, F520, SG001, APL-502, Kosiberimab, Rhodapolimab, GS-4224, INCB086550, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, MAX-10181, RC98, BION-004, AM0001, CB201, ENUM244C8, The anti-PD-1 antagonist antibody is selected from, but is not limited to, ENUM388D4, 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 MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab), but is not limited to these. PD-1 / PD-L1 antagonists include known antibodies and in-house developed antibodies.

[0061] The terms “operatably linked” and “operatably linked” refer to juxtaposing two or more biological sequences of interest with or without spacers or linkers in a relationship that enables them to function as intended. When used in relation to polypeptides, it is intended to mean that the polypeptide sequence is linked in such a way that the linked product will have its intended biological function. For example, an antibody variable region may be operatably linked to a constant region to yield a stable product with antigen-binding activity. An antigen-binding moiety containing two amino acid chains (such as an antibody Fab) can be said to be “operatably linked” to an Fc region if one chain of that moiety is linked to one chain of the Fc region in such a way that both of their respective functions can be performed. The term can also be used in relation to polynucleotides. For example, when a polynucleotide encoding a polypeptide is operatably linked to a control sequence (e.g., a promoter, enhancer, or silencer sequence), it is intended to mean that the polynucleotide sequence is linked in such a way that it enables the controlled expression of the polypeptide from the polynucleotide. The term "operably linked" may be denoted by a hyphen ("-") when used herein to describe domains linked to form a polypeptide, and may refer to direct linking between domains or linking via linkers having a length of 1 to 30 amino acids, e.g., a single amino acid or a series of (G4S)n linkers (n=1 to 5 (1, 2, 3, 4, or 5)).

[0062] In this specification, the term “binding affinity” is used as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or its antigenic portion and an antigen. The binding affinity between two molecules can be quantified by determining the equilibrium dissociation constant (KD). Alternatively, KD can be determined by measuring the reaction rates of complex formation and dissociation using surface plasmon resonance (SPR) (Biacore®) as a non-limiting example. The rate constants corresponding to the binding and dissociation of a monovalent complex are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. The term ka (or kon) refers to the binding rate of an individual antibody-antigen interaction, and the term kd (or koff) refers to the dissociation rate of an individual antibody-antigen interaction. KD is related to ka and kd by the formula KD = kd / ka or koff / kon. The antibody binding reaction rate and binding affinity can be evaluated by standard assays known in the art or by methods described in the Examples section below.

[0063] The term "EC" as used herein 50 The term "IC" is also called "50% effective concentration" and refers to the concentration of a drug, antibody, or toxin that induces an intermediate response between baseline and maximum after a specific exposure time. 50 The term "50% inhibitory concentration," also known as "50% inhibitory concentration," refers to the 50% 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 a biological or biochemical function. In the context of this application, EC 50 and IC 50 It is expressed in units of "nM" or "M".

[0064] 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 degree. In some embodiments, the bispecific polypeptide complexes disclosed herein have IC50 concentrations of ≤1 nM, ≤0.8 nM, ≤0.6 nM, ≤0.4 nM, or ≤0.3 nM. 50This blocks the binding of human TIGIT to PVR. In some embodiments, the bispecific polypeptide complex disclosed herein has an IC50 of 1 nM, 0.9 nM, or 0.8 nM. 50 This blocks the binding of human PVRIG to PVRL2.

[0065] As used herein, the term “isolated” refers to a state obtained from its natural state by artificial means. Where a particular “isolated” substance or component exists in nature, this is made possible by alteration of its natural environment, or by isolation of the substance from its natural environment, or both. For example, certain non-isolated polynucleotides or polypeptides exist naturally within certain animal organisms, and a high-purity version of the same polynucleotide or polypeptide isolated from such a natural state is called an isolated polynucleotide or polypeptide. The term “isolated” does not exclude any artificial or synthetic substances or other impurities that do not affect the activity of the isolated substance.

[0066] As used herein, the term “vector” refers to a nucleic acid vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it is capable of expressing a protein encoded by the inserted polynucleotide. A vector can express its genetic material elements in a host cell by transformation, transduction, or transfection of the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), phages such as lambda phages or M13 phages, 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 papovaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but is not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may contain an origin of replication.

[0067] As used herein, the term “host cell” refers to a cell line that can be manipulated to produce the protein, protein fragment, or peptide of interest. Examples of host cells include, but are not limited to, cultured mammalian cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, and YB2 / 0; human tissue or hybridoma cells; yeast cells; insect cells; and cells contained within transgenic animals or cultured tissues. This term encompasses not only individual target cells but also their offspring. Such offspring may not be identical to their parent cells due to mutations or environmental influences, but they are still included within the scope of the term “host cell.”

[0068] 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, determined by aligning and comparing their sequences. “Identity percentage” means the percentage of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the smallest size of the compared molecules. For these calculations, any gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., “algorithm”). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides 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.

[0069] 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 properties of an antigen that stimulate specific immune cells to be activated, proliferate, and differentiate to ultimately produce immunological effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response in which antibodies or sensitized T lymphocytes are formed in the organism’s immune system after the organism is stimulated by the antigen. Immunogenicity is the most important property of an antigen. Whether an antigen can successfully induce the production of an immune response in a host depends on three factors: the properties of the antigen, the host’s reactivity, and the means of immunization.

[0070] 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. Several 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, Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, and Chu et al., 1981, Gene 13:197. In a particular embodiment of the present invention, the human TIGIT gene was transfected into 293F cells.

[0071] As used herein, the terms “SPR” or “Surface Plasmon Resonance” refer to, and include, optical phenomena that enable real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix using systems such as the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, New Jersey). For further explanation, see Example 5 and Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26, Jonsson, U., et al. (1991) Biotechniques 11:620-627, Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131, and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0072] As used herein, the terms “fluorescence-activated cell sorting” or “FACS” refer to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of living cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell (FlowMetric. “Sorting Out Fluorescence Activated Cell Sorting”. Retrieved 2017-11-09). Instruments for performing FACS are known to those skilled in the art and are generally commercially available. Examples of such instruments include the FACS Star Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, California), the Epics C from Coulter Epics Division (Hyalia, Florida), and the MoFlo from Cytomation (Colorado Springs, Colorado).

[0073] 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 mammalian species. This term does not necessarily indicate that the subject has been diagnosed with a specific disease and typically refers to an individual under medical supervision.

[0074] As used herein in the context of preventing a condition, the terms “prevent,” “prevent,” or “prevention” generally mean preventing or delaying the onset of a disease, or preventing the manifestation of its clinical or subclinical symptoms in an object (whether human or animal), for example, preventing the disease from developing in an object that is predisposed to a condition or disease but has not yet been diagnosed as having it.

[0075] As used herein in the context of treating a condition, the terms “treatment,” “treating,” or “treated” generally refer to treatments and therapies, whether in humans or animals, that achieve any desired therapeutic effect, such as inhibition of the progression of a condition, including but not limited to a reduction in the rate of progression, cessation of progression, regression of the condition, improvement of the condition, and cure of the condition. In the case of cancer, “treating” may mean weakening or slowing the growth, proliferation, or metastasis of a tumor or malignant cells, or any combination thereof.

[0076] As used herein, the term “effective dose” refers to the amount of an active compound, or a material, composition, or dosage form containing an active compound, that is effective in producing some desired therapeutic effect commensurate with a reasonable benefit-to-risk ratio when administered according to a desired therapeutic regimen. For example, when used in connection with the treatment of a disease or condition, “effective dose” refers to an amount or concentration of antibody or its antigen-binding moiety that is effective in treating the disease or condition.

[0077] As used herein, the term “pharmaceutically acceptable” means that its vehicle, diluent, excipient and / or salt is chemically and / or physically compatible with the other components of the formulation and physiologically compatible with the recipient.

[0078] As used herein, the term “pharmaceutically acceptable carriers and / or excipients” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and activator, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences, Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are 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.

[0079] As used herein, the term “adjuvant” refers to a nonspecific immunostimulant that, when delivered to an organism with an antigen or prior to the antigen, can enhance the immune response to an antigen or alter the type of immune response in the organism. Various adjuvants exist, including, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund’s adjuvants (e.g., complete Freund’s adjuvant and incomplete Freund’s adjuvant), Corynebacterium parvum, lipopolysaccharides, and cytokines. Freund’s adjuvants are currently the most commonly used adjuvants in animal studies. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0080] Polypeptide complex targeting TIGIT and PVRIG The polypeptide complexes provided herein comprise a bispecific antibody and its antigen-binding moiety. As used herein, the term “polypeptide complex” may be used interchangeably with “antibody.” In some embodiments, the bispecific antibody and its antigen-binding moiety have a first specificity for TIGIT (e.g., human, cynomolgus monkey, and mouse TIGIT) and a second specificity for PVRIG (e.g., human and cynomolgus monkey PVRIG). Such antibodies may be referred herein, for example, to “anti-TIGIT / anti-PVRIG,” “anti-PVRIG / TIGIT,” “anti-PVRIGxTIGIT,” or “PVRIGxTIGIT” bispecific antibodies, or other similar terms.

[0081] In some embodiments, the bispecific antibodies described herein include 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. When constructing a bispecific antibody, the first and second antigen-binding moieties may be in Fab, scFv, or VHH format, taking into consideration the stability, expression level, binding ability, and other functions of the assembled antibody. For example, the TIGIT-binding moiety may be in Fab format and the PVRIG-binding moiety may be in scFv format. Alternatively, the PVRIG-binding moiety may be in Fab format and the TIGIT-binding moiety may be in scFv format. In some embodiments, both the TIGIT-binding moiety and the PVRIG-binding moiety are in Fab format.

[0082] In some embodiments, the bispecific antibodies disclosed herein include multiple antigen-binding moieties that specifically bind to PVRIG and / or multiple antigen-binding moieties that specifically bind to TIGIT. Typically, in the case of a bispecific antibody, the multiple antigen-binding moieties either have the same variable region (and therefore target the same antigen / epitope) or the variable region and constant region (if any) are identical. For example, an antibody may include two identical PVRIG-binding moieties and one TIGIT-binding moiety, or one PVRIG-binding moiety and two identical TIGIT-binding moieties, or two identical PVRIG-binding moieties and two identical TIGIT-binding moieties. Furthermore, if two PVRIG-binding moieties or two TIGIT-binding moieties are present, these moieties may adopt the following formats: (i) the TIGIT-binding moiety is Fab and the PVRIG-binding moiety is scFv; (ii) the TIGIT-binding moiety is scFv and the PVRIG-binding moiety is Fab; or (iii) both the TIGIT-binding moiety and the PVRIG-binding moiety are Fab.

[0083] In some specific embodiments, the TIGIT binding portion is a Fab format comprising a first heavy chain variable domain (VH1) (VH1-CH1) operably linked to the antibody heavy chain CH1 domain and a first light chain variable domain (VL1) (VL1-CL) operably linked to the antibody light chain constant (CL) domain, and the PVRIG binding portion is an scFv format comprising a second heavy chain variable domain (VH2) (VH2-VL2) operably linked to a second light chain variable domain (VL2). The PVRIG-binding scFv may be located at the N-terminus or C-terminus of the heavy or light chain of the bispecific polypeptide complex, and in the scFv, VH2 may be at the N-terminus of VL2, or vice versa.

[0084] In some specific embodiments, the TIGIT binding portion is an scFv format comprising a first VH (VH1-VL1) operably linked to a first VL, and the PVRIG binding portion is a Fab format comprising a second VH (VH2-CH1) operably linked to the antibody heavy chain CH1 domain and a second VL (VL2-CL) operably linked to the antibody light chain constant (CL) domain. The TIGIT-binding scFv may 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 may be at the N-terminus of VL1, or vice versa.

[0085] As an example, the bispecific polypeptide complex disclosed herein may comprise two heavy chains and two light chains, where the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, and from the N-terminus to the C-terminus, (a) The first heavy chain and the second heavy chain each contain operably linked domains such as VH1-CH1-hinge-Fc-scFv, and the first light chain and the second light chain each contain operably linked domains such as VL1-CL, or (b) The first heavy chain and the second heavy chain each contain domains that are operably linked in the form scFv-VH1-CH1-hinge-Fc, and the first light chain and the second light chain each contain domains that are operably linked in the form VL1-CL, VH1-CH1 and VL1-CL originate from the TIGIT binding site, while scFv (VH2-VL2 or VL2-VH2 from the N-terminus to the C-terminus) originates from the PVRIG binding site.

[0086] As another example, the bispecific polypeptide complex disclosed herein may comprise two heavy chains and two light chains, where the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, (a) The first heavy chain and the second heavy chain each contain operably linked domains such as VH2-CH1-hinge-Fc-scFv, and the first light chain and the second light chain each contain operably linked domains such as VL2-CL, or (b) The first heavy chain and the second heavy chain each contain domains that are operably linked in the form of scFv-VH2-CH1-hinge-Fc, and the first light chain and the second light chain each contain domains that are operably linked in the form of VL2-CL, scFv (VH1-VL1 or VL1-VH1 from the N-terminus to the C-terminus) originates from the TIGIT binding site, while VH2-CH1 and VL2-CL originate from the PVRIG binding site.

[0087] In some specific embodiments, the TIGIT binding portion is a Fab format comprising a first VH (VH1-CH1) operably linked to the antibody heavy chain CH1 domain and a first VL (VL1-CL) operably linked to the antibody light chain constant (CL) domain, and the PVRIG binding portion is also a Fab format comprising a second heavy chain variable domain (VH2-CH1) operably linked to the antibody heavy chain CH1 domain and a second VL (VL2-CL) operably linked to the antibody light chain constant (CL) domain. The TIGIT binding portion may be located at the N-terminus of the Fc region and the PVRIG binding portion 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 at the N-terminus of the Fc region. In other embodiments, 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, TIGIT-binding Fab and PVRIG-binding Fab can be distinguished by substituting CH1 and CL of the TIGIT-binding or PVRIG-binding moiety with a pair of TCR constant regions. Therefore, in some embodiments, the CH1 domain is substituted with a first TCR constant region (C1, i.e., wild-type or engineered TCR beta-chain constant region), and the CL domain is substituted with a second TCR constant region (C2, i.e., wild-type or engineered TCR alpha-chain constant region). Alternatively, they may be swapped to form the TCR beta-chain constant region in the light chain and the TCR alpha-chain constant region in the heavy chain.

[0088] The T cell receptor (TCR) is a heterodimeric T cell surface protein belonging to the immunoglobulin superfamily, similar to a semi-antibody having a single heavy chain and a single light chain. Natural TCRs have an extracellular portion, a transmembrane portion, and an intracellular portion. The extracellular domain of the TCR has a membrane-proximal constant region and a membrane-distal variable region. The introduction of the TCR constant region to replace commonly used CH1 and CL domains has been shown to increase the stability and expression levels of the resulting antibody format. A detailed description of the usefulness of the TCR constant region in assembling two parental antibodies into a bispecific molecule with desired titer and functionality is disclosed in International Publication No. 2019 / 057122, which is incorporated herein by reference in its entirety. Substitution with the TCR constant region results in a chimeric Fab with a unique light-heavy chain interface orthogonal to that of a normal antibody Fab. The assembly of chimeric Fabs of different formats and normal Fabs can create a variety of bispecific molecules with different structures and titers.

[0089] A pair of TCR constant regions in a chimeric Fab includes a TCR alpha constant region (wild-type or preferably engineered) in the light chain and a TCR beta constant region (wild-type or preferably engineered) in the heavy chain. In some embodiments, the chimeric Fab includes a first TCR constant region and a second TCR constant region linked via a non-natural interchain disulfide bond. The TCR constant regions may be manipulated to introduce a non-natural disulfide bond at the light-heavy chain interface. In some other embodiments, the chimeric Fab includes a pair of wild-type TCR constant regions, e.g., wild-type human TCR beta and alpha constant regions.

[0090] The sequences of the constant regions of the wild-type human TCR beta and alpha chains can be found at NCBI accession numbers A0A5B9 (www.uniprot.org / uniprot / A0A5B9) and P01848 (www.uniprot.org / uniprot / P01848). The pair of TCR constant regions for constructing bispecific antibodies described herein are derived from wild-type TCR constant regions and have one or more substitutions, additions, or deletions of one or more amino acids. The bispecific antibody may also include the engineered TCR beta chain constant region and the engineered TCR alpha chain constant region (referred to herein as C1 and C2). As illustrated in this application, a bispecific antibody may comprise an engineering TCR beta chain constant region (C1) having the sequence shown in SEQ ID NO: 21 (LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALQDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGR) and an engineering TCR alpha chain constant region (C2) having the sequence shown in SEQ ID NO: 22 (PDIQNPDCAVYQLRDSKSSDKSVCLFTDFDSQTQVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSQKSDFACANAFQNSIIPECTFFPS). Multiple TCR constant region variants for constructing a bispecific antibody format are disclosed in PCT / CN2021 / 072601, the full contents of which are incorporated herein by reference.

[0091] As an example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, where the TIGIT binding portion is in Fab format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first and second heavy chains each contain operably linked domains such as VH1-C1-VH2-CH1-hinge-Fc or VH2-CH1-VH1-C1-hinge-Fc (where VH1-C1 originates from the TIGIT binding site and VH2-CH1 originates from the PVRIG binding site), the two light chains contain operably linked domains such as VL1-C2 (where VL1-C2 originates from the TIGIT binding site), and the other two light chains contain operably linked domains such as VL2-CL (where VL2-CL originates from the PVRIG binding site).

[0092] As another example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, where the TIGIT binding portion is in Fab format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first and second heavy chains each contain operably linked domains such as VH1-C1-hinge-Fc-VH2-CH1 or VH2-CH1-hinge-Fc-VH1-C1 (where VH1-C1 originates from the TIGIT binding site and VH2-CH1 originates from the PVRIG binding site), the two light chains contain operably linked domains such as VL1-C2 (where VL1-C2 originates from the TIGIT binding site), and the other two light chains contain operably linked domains such as VL2-CL (where VL2-CL originates from the PVRIG binding site).

[0093] As another example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, where the TIGIT binding portion is in Fab format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first and second heavy chains each contain operably linked domains such as VH1-CH1-VH2-C1-hinge-Fc or VH2-C1-VH1-CH1-hinge-Fc (where VH1-CH1 originates from the TIGIT binding site and VH2-C1 originates from the PVRIG binding site), the two light chains contain operably linked domains such as VL1-CL (where VL1-CL originates from the TIGIT binding site), and the other two light chains contain operably linked domains such as VL2-C2 (where VL2-C2 originates from the PVRIG binding site).

[0094] As yet another example, the bispecific polypeptide complex disclosed herein comprises two heavy chains and four light chains, where the TIGIT binding portion is in Fab format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first and second heavy chains each contain operably linked domains such as VH1-CH1-hinge-Fc-VH2-C1 or VH2-C1-hinge-Fc-VH1-CH1 (where VH1-CH1 originates from the TIGIT binding site and VH2-C1 originates from the PVRIG binding site), the two light chains contain operably linked domains such as VL1-CL (where VL1-CL originates from the TIGIT binding site), and the other two light chains contain operably linked domains such as VL2-C2 (where VL2-C2 originates from the PVRIG binding site).

[0095] In some embodiments, the bispecific antibodies disclosed herein include one TIGIT binding moiety and two PVRIG binding moieties per antibody, or two TIGIT binding moieties and one PVRIG binding moiety per antibody. These moieties may be in Fab or scFv format.

[0096] As an example, the bispecific polypeptide complex described herein comprises two heavy chains and two light chains, where the TIGIT binding portion is in Fab format and the PVRIG binding portion is in scFv format, and from the N-terminus to the C-terminus, The first heavy chain includes operably linked domains such as VH1-CH1-hinge-Fc-scFv or scFv-VH1-CH1-hinge-Fc, the second heavy chain includes operably linked domains such as VH1-CH1-hinge-Fc, and the two light chains include operably linked domains such as VL1-CL. VH1-CH1 and VL1-CL originate from the TIGIT binding site, while scFv (VH2-VL2 or VL2-VH2 from the N-terminus to the C-terminus) originates from the PVRIG binding site.

[0097] As another example, the bispecific polypeptide complex described herein comprises two heavy chains and two light chains, where the TIGIT binding portion is in scFv format and the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first heavy chain includes operably linked domains such as VH2-CH1-hinge-Fc-scFv or scFv-VH2-CH1-hinge-Fc, the second heavy chain includes operably linked domains such as VH2-CH1-hinge-Fc, and the two light chains include operably linked domains such as VL2-CL. VH2-CH1 and VL2-CL originate from the PVRIG binding site, while scFv (VH1-VL1 or VL1-VH1 from the N-terminus to the C-terminus) originates from the TIGIT binding site.

[0098] Depending on the bispecificity format and / or numbering requirements, each numbered sequence disclosed herein, for example, the first or the second, may be different. For example, the first VH may be numbered as the second VH, and the first VL may be numbered as the second VL. As another example, depending on preference and / or convenience, the second antigen-binding portion may be numbered as the first antigen-binding portion, and the first antigen-binding portion may be numbered as the second antigen-binding portion.

[0099] CDR and variable regions of bispecific polypeptide complexes In some embodiments, the bispecific polypeptide complex or its antigen-binding moiety comprises a first antigen-binding moiety that specifically binds to TIGIT (preferably human TIGIT) and a second antigen-binding moiety that specifically binds 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 a known antibody already developed or a novel antibody. "Derived from" generally means, as used herein, that the antigen-binding moiety comprises the CDR sequence of the parent antibody or a highly homologous CDR sequence, preferably comprising the variable region of the parent antibody. The antigen-binding moiety may also comprise variants of the CDR sequence of the parent antibody that retain 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.

[0100] Specifically, in the bispecific antibodies exemplified herein, the TIGIT binding moiety is A) (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having the addition, deletion, and / or substitution of 1, 2, or 3 or fewer amino acids compared to SEQ ID NO: 1, (ii) HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having 1, 2, or 3 or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 2, and (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having 1, 2, or 3 or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 3. One or more heavy chain CDRs (HCDRs) selected from the group consisting of, B) (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having the addition, deletion, and / or substitution of 1, 2, or 3 or fewer amino acids compared to SEQ ID NO: 4, (ii) LCDR2 including the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having 1, 2, or 3 or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 5, (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 6. One or more light chain CDRs (LCDRs) selected from the group consisting of, or C) One or more HCDRs from A) and B) One or more LCDRs Includes, The PVRIG bond portion is A') (i) HCDR1 comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 7, (ii) HCDR2 including the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 8, (iii) HCDR3 containing the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having 1, 2, or 3 or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 9. One or more HCDRs selected from the group consisting of, B') (i) LCDR1 containing the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 10, (ii) LCDR2 including the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having the addition, deletion, and / or substitution of 1, 2, or 3 or fewer amino acids compared to SEQ ID NO: 11, and (iii) LCDR3 containing the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having one, two, or three or fewer amino acid additions, deletions, and / or substitutions compared to SEQ ID NO: 12. One or more LCDRs selected from the group consisting of, C') A') one or more HCDRs and B') one or more LCDRs Includes.

[0101] In some embodiments, the CDR of the anti-TIGIT moiety is identified according to the Contact definition, and the CDR of the anti-PVRIG moiety is identified according to the IMGT / Kabat definition. Variable regions and CDRs in antibody sequences can be identified according to general rules developed in the art, or by aligning the sequence against a database of known variable regions. CDRs are described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), Kabat et al., USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes duplicates or subsets of amino acid residues when compared to one another. However, the application of any definition used to represent the CDRs of antibodies disclosed herein is intended to be within the scope of this application. The CDRs shown in Table A below are defined by Contact, or by the Kabat and IMGT numbering systems, but CDRs may be defined using Chothia, MacCallum, and other methods known in the art. Methods for identifying 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 available on the "Abysis" website (www.bioinf.org.uk / abs, maintained by AC Martin of the Department of Biochemistry and Molecular Biology, University College London, UK) and the VBASE2 website (www.vbase2.org, described in Retter et al., Nucl. Acids Res., 33 (Database issue): D671-D674 (2005)), and can be accessed through these sites. Preferably, sequences are analyzed using the Abysis database, which integrates sequence data from Kabat, IMGT, and the Protein Data Bank (PDB) with structural data from the PDB. See the chapter on Protein Sequence and Structure Analysis of Antibody Variable Domains in the Antibody Engineering Lab Manual by Dr. Andrew CRMartin (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg, ISBN-13: 978-3540413547, also available on the website bioinforg.uk / abs). The Abysis database website further contains general rules developed for identifying CDRs, which can be used in accordance with the teachings herein.

[0102] In some specific embodiments, the TIGIT bond region includes a heavy chain variable region (VH) and a light chain variable region (VL). (a) VH includes HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, and HCDR3 shown in SEQ ID NO: 3. (b) VL includes LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0103] In some specific embodiments, the PVRIG binding region includes a heavy chain variable region (VH) and a light chain variable region (VL). (a) VH includes HCDR1 shown in SEQ ID NO: 7, HCDR2 shown in SEQ ID NO: 8, and HCDR3 shown in SEQ ID NO: 9. (b) VL includes LCDR1 shown in sequence number 10, LCDR2 shown in sequence number 11, and LCDR3 shown in sequence number 12.

[0104] In some embodiments, the bispecific antibody or its antigen-binding moiety includes a first antigen-binding moiety that specifically binds to TIGIT, and the first antigen-binding moiety is (A) (i) Contains the amino acid sequence of SEQ ID NO: 13, (ii) Containing at least 85%, 90%, or 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: 13, or (iii) 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 acid additions, deletions and / or substitutions compared to SEQ ID NO: 13 Heavy chain variable region, and / or (B) (i) Contains the amino acid sequence of SEQ ID NO: 14, (ii) Containing 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 (iii) 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 acid additions, deletions and / or substitutions compared to SEQ ID NO: 14 Includes a variable light chain region.

[0105] In some further embodiments, the bispecific antibody or its antigen-binding moiety includes a second antigen-binding moiety that specifically binds to PVRIG, and the second antigen-binding moiety is (A) (i) Contains the amino acid sequence of SEQ ID NO: 15, (ii) Containing at least 85%, 90%, or 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: 15, or (iii) 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 acid additions, deletions and / or substitutions compared to SEQ ID NO: 15 Heavy chain variable region, and / or (B) (i) Contains the amino acid sequence of SEQ ID NO: 16, (ii) Containing 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 (iii) 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 acid additions, deletions and / or substitutions compared to SEQ ID NO: 16 Includes a variable light chain region.

[0106] Preferably, an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with SEQ ID NOs. 13, 14, 15, or 16 has the same CDR sequence as SEQ ID NOs. 13, 14, 15, or 16, and amino acid changes occur only in the framework region. The percentage of identity between two amino acid sequences can be determined using the E. Meyers and W. Miller algorithm (Comput.Appl.Biosci.,4:11-17(1988)) incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J.Mol.Biol.48:444-453(1970)), incorporated into the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum62 matrix or the PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0107] Additionally or alternatively, the protein sequences of this disclosure can be used as "query sequences" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J.MoI. Biol. 215:403-10. The BLAST protein search 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. To obtain gapped alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0108] In some specific embodiments, the heavy chain variable region of the TIGIT binding portion consists of the amino acid sequence of SEQ ID NO: 13, the light chain variable region of the TIGIT binding portion consists of the amino acid sequence of SEQ ID NO: 14, and / or the heavy chain variable region of the PVRIG binding portion consists of the amino acid sequence of SEQ ID NO: 15, and the light chain variable region of the PVRIG binding portion consists of the amino acid sequence of SEQ ID NO: 16.

[0109] In other embodiments, the amino acid sequences of the heavy chain variable region and / or light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least 95%, 96%, 97%, 98%, or 99% identical to the respective sequences described above.

[0110] In some further embodiments, a bispecific antibody or its antigen-binding moiety may include conservative amino acid substitutions or modifications in the variable regions of the heavy and / or light chains. In the Art, it is understood that certain conservative sequence modifications can be made without removing antigen binding. For example, see Brummell et al. (1993) Biochem 32:1180-8, de Wildt 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 and O' 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.

[0111] As described above, the term "conservative substitution" refers to amino acid substitutions that will not adversely affect or alter the essential properties of a protein / polypeptide, including its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis or 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, 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)). Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charging 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 (e.g., 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. Methods for identifying 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) (these are incorporated herein by reference)).

[0112] Antigen binding of bispecific polypeptide complexes Preferably, the bispecific polypeptide complex of the present disclosure can bind to human TIGIT and PVRIG. Binding of the antibody to TIGIT and PVRIG can be evaluated using one or more techniques well established in the art, such as ELISA or FACS, which measure the binding of the antibody to soluble TIGIT / PVRIG protein or TIGIT / PVRIG protein expressed on the cell surface, respectively. For example, the antibody can be tested by a flow cytometry assay that reacts the antibody with a cell line expressing human TIGIT or human PVRIG, such as a CHO cell transfected to express TIGIT or PVRIG on the cell surface, or a TIGIT- or PVRIG-positive cell line, or a TIGIT and PVRIG double-positive cell line.

[0113] Additionally or alternatively, binding of the antibody, such as the binding reaction rate (e.g., KD value), can be tested in a BIAcore binding assay. For example, the bispecific antibody of the present disclosure has a K -10 of 1×10 D M or less, a K -11 of 8×10 D M or less, a K -11 of 6×10 D M or less, a K -11 of 4×10 D M or less, or a K -11 of 2×10 D M or less when binding to human TIGIT protein, and a K -9 of 5×10 D M or less, a K -9 of 4×10 D M or less, or a K -9 of 3×10 D M or less when binding to human PVRIG protein.

[0114] Functionality of BsAb 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. (a) Specifically binds to human, cynomolgus monkey, and mouse TIGIT, (b) Specifically binds to human and cynomolgus monkey PVRIG, (c) It must be possible to be coupled to both TIGIT and PVRIG simultaneously. (d) Efficiently block the coupling between PVRIG and PVRL2 and the coupling between TIGIT and PVR. (e) It does not have cross-activity with paralog proteins of TIGIT and PVRIG. (f) In reporter gene assays, NK cell killing assays, and T cell activation assays, the antibodies should be shown to be superior to those of control antibodies (WBP364-BMK1 and WBPT117-BMK1) and to combinations thereof. (g) Possessing good potential for antibody development, including thermal stability, solubility, hydrophobicity, and stress stability. (h) Having an acceptable pharmacokinetic profile in monkeys, (i) To demonstrate a significant antitumor effect in the CT26 mouse model.

[0115] 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 the parent antibody from which the bispecific antibody is derived and constructed. In some embodiments, the control antibody is W3642 and / or WT1175.

[0116] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher binding affinity to TIGIT compared to monospecific anti-TIGIT antibodies or other anti-TIGIT / PVRIG bispecific antibodies. In some embodiments, the bispecific polypeptide complexes disclosed herein have a binding affinity to TIGIT that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than monospecific anti-TIGIT antibodies, as measured by SPR or FACS.

[0117] In some embodiments, the bispecific polypeptide complexes disclosed herein have a higher or equivalent binding affinity to PVRIG compared to monospecific anti-PVRIG antibodies or other anti-TIGIT / PVRIG bispecific antibodies. In some embodiments, the bispecific polypeptide complexes disclosed herein have a binding affinity to PVRIG that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% higher than monospecific anti-PVRIG antibodies or other anti-TIGIT / PVRIG bispecific antibodies, as measured by FACS.

[0118] The anti-TIGIT / PVRIG antibodies disclosed herein can inhibit the interaction between TIGIT and its ligand PVR (CD155), and the interaction between PVRIG and PVRL2 (CD112). By blocking these signaling pathways, functional responses by T cells to antigen stimulation (e.g., proliferation, cytokine production, target cell killing) can be restored from a dysfunctional state.

[0119] The ability of an antibody to inhibit the interaction between an antigen and its ligand may be evaluated in a binding assay by measuring, for example, whether the physical interaction between TIGIT and PVR is reduced. In some embodiments, the binding assay is a competitive binding assay. The assay may be carried out in various formats, including, but is not limited to, ELISA assays, flow cytometry, surface plasmon resonance (SPR) assays (e.g., Biacore®), or biolayer interferometry (e.g., ForteBio Octet®).

[0120] Multiple blocking of TIGIT, PVRIG, and PD-1 / PD-L1 can reverse immunosuppression. The bispecific antibodies of this disclosure may 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).

[0121] In some preferred embodiments, the antibodies of this disclosure may be used in combination with additional therapeutic agents, such as anticancer agents, including anticancer antibodies and chemotherapeutic agents. The additional therapeutic agents may be antagonists or inhibitors of T cell co-suppressive molecules, agonists of T cell co-activating molecules, or immunostimulatory cytokines.

[0122] In some embodiments, the additional therapeutic agent is an antibody that binds to a protein 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, the additional therapeutic agent is selected from anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD47M antibody, anti-CSF1R antibody, anti-TLR antibody, anti-RIGI antibody, anti-TAM receptor kinase antibody, anti-NKG2A antibody, anti-CD25 antibody, anti-NKG2D antibody, anti-GD2 antibody, anti-PDGFR-a antibody, anti-SLAMF7 antibody, anti-VEGF antibody, anti-CD20 antibody, anti-cCLB8 antibody, anti-KIR antibody, and anti-CD52 antibody. In some embodiments, additional therapeutic agents are selected from Hul4.18K322A, Hu3F8, dinituximab, olaratumab, elotuzumab, ramucirumab, bevacizumab, rituximab, silutuximab, lirirumab, and alemtuzumab.

[0123] Fc area This disclosure provides a polypeptide complex comprising a TIGIT binding moiety, a PVRIG binding moiety, and a dimerization pair comprising two dimerization domains that facilitate the assembly of at least two polypeptide chains. The dimerization pair may include, for example, but is not limited to, immunoglobulin constant regions (e.g., Fc, CH2 and / or CH3 domains of heavy-chain immunoglobulin).

[0124] In certain embodiments, the bispecific antibodies and their antigen-binding moieties provided herein may include an immunoglobulin constant region comprising an Fc region, such as a human IgG1, IgG2, IgG3, or IgG4 Fc region (natural type or a variant thereof), and optionally a hinge region. 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 or at least about 90% homology, e.g., at least about 95% homology, with the natural sequence Fc region. In some embodiments, the Fc region is a human IgG4 Fc region, such as a wild-type Fc region or an Fc variant. In certain embodiments, the bispecific polypeptide complexes disclosed herein include a variant Fc region having one or more amino acid modifications (e.g., Leu234Ala / Leu235Ala or LALA, as numbered in EU, such as Kabat et al.) that alter antibody-dependent cell-mediated cytotoxicity (ADCC) or other effector function. In some embodiments, the Fc region may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) that result in a modified Fc region in which the binding interaction between Fc and FcRn or FcγR is altered.

[0125] In some embodiments, the Fc region may include one or more amino acid changes (e.g., insertions, deletions, or substitutions) to obtain a desired functionality, such as a "knob-into-hole" structure that promotes heterodimerization (if necessary).

[0126] As used herein, the term “knob-into-hole” refers to manipulating the CH3 domain of the antibody Fc region to create either a “knob” or a “hole” in each heavy chain to promote heterodimerization. Knobs can be obtained by substituting a small amino acid residue with a large amino acid residue in the first CH2 / CH3 polypeptide, while holes can be obtained by substituting a large residue with a small residue. For details on mutation sites for knob-into-hole, see Spiess et al., 2015 and Brinkmann et al., 2017 cited above. Generally, a “knob” is created by substituting T366 with a bulky residue W on one heavy chain, according to EU numbering such as Kabat et al., while the corresponding “hole” is created by a triple mutation of T366S, L368A, and Y407V on the other heavy chain. In some embodiments, the heavy chain of a bispecific antibody containing C1 has a “hole” structure, while the heavy chain containing C2 has a “knob” structure. Alternatively, the heavy chain of a bispecific antibody containing C1 has a "knob" structure, while the heavy chain containing C2 has a "hole" structure.

[0127] In certain embodiments, the Fc region of the bispecific antibody herein is an IgG4 Fc region containing the S228P mutation (as numbered in EU, such as Kabat et al.) which prevents Fab arm exchange and stabilizes the IgG4 molecule. In certain embodiments, the Fc region is an IgG1 Fc region containing LALA mutations, namely L234A and L235A (as numbered in EU, such as Kabat et al.). LALA mutations are perhaps the most commonly used mutations because they interfere with antibody effector function, for example, by eliminating Fc binding to specific FcγR and reducing PBMC and monocyte-mediated ADCC activity.

[0128] In some embodiments, the Fc region may be manipulated to prevent glycosylation, extend its half-life, or modulate receptor binding or effector function. Examples of mutations are discussed in Saunders KO (Front.Immunol.10:1296,2019 (the full content of which is incorporated herein by reference)) and include, for example, mutations in asparagine 297 (e.g., N297 with EU numbering, as in Kabat et al.).

[0129] In exemplary embodiments, the polypeptide complexes disclosed herein may have mutant constant regions or Fc regions having sequences that are 80% to 99% identical to the sequences of the natural IgG1, IgG2, IgG3, or IgG4 constant regions or Fc regions. For example, mutant Fc regions that are 85% to 99%, 90% to 99%, or 95% to 99% identical to the natural IgG1, IgG2, IgG3, or IgG4 Fc regions are included in the present invention.

[0130] The terms "EU numbering system" or "EU index" are generally used to refer to residues in the constant region of immunoglobulin heavy chains (e.g., the EU index reported by Kabat et al. above). "Kabat-like EU numbering" or "Kabat-like EU index" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, references to residue numbers in the constant domain of an antibody mean residue numbering according to the EU numbering system.

[0131] Nucleic acid molecules encoding antibodies in this disclosure In some embodiments, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding the heavy chain variable region and / or light chain variable region of a bispecific antibody disclosed herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the heavy chain of a bispecific antibody disclosed herein. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the light chain of a bispecific antibody disclosed herein.

[0132] The nucleic acids of this disclosure can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice possessing human immunoglobulin genes), the cDNA encoding the light and heavy chains of the antibodies produced by the hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from immunoglobulin gene libraries (e.g., using phage display techniques), the nucleic acids encoding such antibodies can be recovered from the gene library.

[0133] Isolated nucleic acids encoding the VH region can be converted into full-length heavy chain genes by operably ligating the VH-encoding nucleic acid to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991) cited above), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant regions may be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant regions, but are preferably IgG1 or IgG4 constant regions.

[0134] Isolated nucleic acids encoding the VL region can be converted into full-length light chain genes (and Fab light chain genes) by operably ligating the VL-encoding DNA to another DNA molecule encoding the light chain constant region CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al. cited above), and DNA fragments containing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa constant region or a lambda constant region.

[0135] Once DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques to convert, for example, a variable region gene into a full-length antibody chain gene, a Fab fragment gene, or an scFv gene. In these manipulations, the DNA fragment encoding VL or VH is operably ligated to another DNA fragment encoding another protein, such as an antibody constant region or a mobile linker. The term "operably ligated" as used in this context is intended to mean that the two DNA fragments are ligated in such a way that the amino acid sequences encoded by these two DNA fragments remain in frame.

[0136] In some embodiments, the present disclosure relates to isolated nucleic acid molecules comprising a heavy chain variable region or a nucleic acid sequence encoding a heavy chain (of the TIGIT-binding or PVRIG-binding portion) of a bispecific antibody disclosed herein.

[0137] In some specific embodiments, isolated nucleic acid molecules are (A) Nucleic acid sequences encoding the amino acid sequences shown in SEQ ID NOs: 13, 15, 17, or 19 (B) A nucleic acid sequence hybridized to the complementary strand of the nucleic acid sequence of (A) under high stringency conditions, or (C) A nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the nucleic acid sequence of (A). Includes.

[0138] In some embodiments, the present disclosure relates to isolated nucleic acid molecules comprising a light chain variable region (of the TIGIT-binding moiety or PVRIG-binding moiety) or a nucleic acid sequence encoding a light chain of a bispecific antibody disclosed herein.

[0139] In some specific embodiments, isolated nucleic acid molecules are (A) Nucleic acid sequences encoding the light chain variable region shown in Sequence ID No. 14, 16, 18, or 20, (B) A nucleic acid sequence hybridized to the complementary strand of the nucleic acid sequence of (A) under high stringency conditions, or (C) A nucleic acid sequence having at least 80% sequence identity (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the nucleic acid sequence of (A). Includes.

[0140] In some embodiments, the identity percentage is due to the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0141] Exemplary high-stringency conditions include hybridization at 45°C in 5×SSPE and 45% formamide, and final washing at 65°C in 0.1×SSC. In the art, it is understood that equivalent stringency conditions can be achieved by varying the temperature and buffer or salt concentration, as described in Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3~6.4.10. Modification of hybridization conditions can be determined empirically or precisely calculated based on probe length and guanosine / cytosine (GC) base pairing ratio. Hybridization conditions can be calculated as described in Sambrook, et al. (Eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47~9.51.

[0142] host cell The host cells disclosed herein may be any cells suitable for expressing the antibodies disclosed herein, such as yeast, bacteria, fungi, plant and animal cells (e.g., mammalian cells). Examples of mammalian host cells for expressing the antibodies disclosed herein include Chinese hamster ovary (CHO cells) (e.g., dhfr CHO cells as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, and used with a DHFR selection marker as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621, etc.), 293F cells, NSO myeloma cells, COS cells and SP2 cells. In particular, when used with NSO myeloma cells, an alternative expression system is the GS gene expression system disclosed in International Publication No. 87 / 04462, International Publication No. 89 / 01036 and European Patent No. 338,841.Additionally, the following cells were used: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL1651), human fetal kidney cell line (293 or 293 cells subcloned for proliferation in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), and human cervical cancer cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor cells (MMT060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68), MRC5 cells, FS4 cells, mouse myeloma cells, e.g., NSO (e.g., RCB0213, 1992, Bio / Technology 10:169) and SP2 / 0 cells (e.g., SP2 / 0-Ag14 cells, ATCC Examples include CRL1581), rat myeloma cells, e.g., YB2 / 0 cells (e.g., YB2 / 3HL.P2.G11.16Ag.20 cells, ATCC CRL1662), PER.C6 cells, and human hepatocellular carcinoma cell lines (Hep G2). CHO cells are one of the cell lines that can be used herein, and 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, they may be modified to lack the ability to fucosylate the expressed proteins.In some embodiments, the host cells herein are selected from CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NSO cells or lymphocytes.

[0143] Suitable prokaryotes for this purpose include bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as the genera Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as Bacillus subtilis and B. licheniformis, Pseudomonas, such as Pseudomonas aeruginosa, and Streptomyces.

[0144] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae or common baker's yeast are among the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains, such as Schizosaccharomyces pombe, Clibellomyces host species, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotlerans, and K. marxianus; Yarrowia (European Patent No. 402,226); Pichia pastris (European Patent No. 183,070); Candida; Trichoderma reesia (European Patent No. 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces Hosts for *Occidentalis* and filamentous fungi, such as those of the genera *Nepeta crassa*, *Penicolus*, *Tripocladium*, and *Aspergillus*, such as *A. nidulans* and *A. niger*, are generally available and useful herein.

[0145] 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 period sufficient to be expressed in the host cells or secreted into the culture medium in which the host cells are cultured. The antibody can be recovered from the culture medium using standard protein purification methods.

[0146] Pharmaceutical composition In some embodiments, this disclosure relates to a pharmaceutical composition comprising at least one antibody or antigen-binding moiety thereof disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a pharmaceutical composition comprising a nucleic acid (DNA or RNA) encoding an antibody disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, this disclosure provides a pharmaceutical composition comprising cells expressing an antibody disclosed herein and a pharmaceutically acceptable carrier.

[0147] Composition components The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical composition of this disclosure may also be administered in combination therapy with another immunostimulant, anticancer agent, antiviral agent or vaccine, etc. Examples of pharmaceutically acceptable carriers include pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspension / dispersant agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, and various combinations or more of other components known in the art.

[0148] Suitable components include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers, such as sugars and cyclodextrins. Suitable antioxidants include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, thioglycolic acid, mercaptosorbitol, butylmethylanisole, butylated hydroxytoluene, and / or propyl gallate. For example, a composition comprising an antibody or antigen-binding fragment of the present disclosure may contain one or more antioxidants (such as methionine) to suppress the oxidation of the antibody or its antigen-binding fragment. Suppression of oxidation can prevent or suppress a decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Accordingly, in some embodiments, the present disclosure provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants (such as methionine). This disclosure further provides various methods by which an antibody or its antigen-binding fragment is mixed with one or more antioxidants (such as methionine) to prevent oxidation of the antibody or its antigen-binding fragment, thereby extending their shelf life and / or increasing their activity.

[0149] Further examples of pharmaceutically acceptable carriers include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as plant-derived fixative oils such as cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents at bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphate buffer or citrate buffer; antioxidants such as sodium bisulfate; and local anesthetics. Examples of excipients include agents such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifiers such as polysorbate 80 (TWEEN®-80), metal ion sequestering agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Antimicrobial agents used as carriers, including phenol or cresol, mercury agents, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride, may be added to the pharmaceutical composition in a multi-dose container. Suitable excipients include, for example, water, physiological saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers, or pharmaceuticals such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.

[0150] Administration, formulation, and dosage The pharmaceutical compositions of this disclosure may be administered in vivo to the target subject as needed by various routes, including, but not limited to, oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, intradermal, topical, transdermal, and intrathecal routes, or otherwise by implantation or inhalation. The compositions may be formulated in the form of solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols, but not limited to these. The appropriate formulation and route of administration may be selected according to the intended use and treatment regimen.

[0151] Formulations suitable for intravenous administration include hard or soft gelatin capsules, pills, coated tablets, elixirs, suspensions, syrups, or inhalants, as well as controlled-release forms thereof.

[0152] 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 further contain other pharmaceutically acceptable components such as antioxidants, buffers, preservatives, stabilizers, bacteriostatic agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the recipient's blood (or other relevant body fluids). Examples of excipients include, for example, water, alcohol, polyols, glycerol, and vegetable oils. Examples of isotonic carriers suitable for use in such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's injection. Similarly, specific dosing regimens, including dosage, timing, and repetition, will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetics (e.g., half-life, clearance rate, etc.).

[0153] The frequency of administration may be determined and adjusted throughout the course of treatment, based on reducing the number of proliferative or tumorigenic cells, maintaining a reduction in such neoplastic cells, reducing the proliferation of neoplastic cells, or delaying the development of metastasis. In some embodiments, the dose administered may be adjusted or attenuated to manage potential side effects and / or toxicity. Alternatively, a sustained-release formulation of the therapeutic composition in question may be appropriate.

[0154] Those skilled in the art will understand that the appropriate dosage may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit with any risks or adverse side effects. The dosage level selected will depend on various factors, but are not limited to, the activity of the specific compound, the route of administration, the timing of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and / or materials used in combination, the severity of the condition, and the patient's species, sex, age, weight, condition, overall health, and medical history. The dosage is generally selected to achieve a local concentration at the site of action that produces the desired effect without causing significant adverse side effects, but the amount of compound and the route of administration are ultimately at the discretion of the physician, veterinarian, or clinician.

[0155] In general, the antibody or its antigen-binding moiety of this disclosure can be administered in a variety of doses. These include approximately 5 μg / kg body weight to approximately 40 mg / kg body weight per dose, approximately 50 μg / kg body weight to approximately 5 mg / kg body weight per dose, and approximately 100 μg / kg body weight to approximately 10 mg / kg body weight per dose. Other ranges include approximately 100 μg / kg body weight to approximately 20 mg / kg body weight per dose, and approximately 0.5 mg / kg body weight to approximately 20 mg / kg body weight per dose. In certain embodiments, the dose is at least approximately 100 μg / kg body weight, at least approximately 250 μg / kg body weight, at least approximately 750 μg / kg body weight, at least approximately 3 mg / kg body weight, at least approximately 5 mg / kg body weight, and at least approximately 10 mg / kg body weight.

[0156] In any case, the antibody or its antigen-binding portion of this disclosure is preferably administered as needed to the target. The frequency of administration can be determined by a person skilled in the art, such as the attending physician, taking into consideration the condition being treated, the age of the target, the severity of the condition, and the general health status of the target.

[0157] In certain preferred embodiments, a therapeutic course comprising the antibody or its antigen-binding moiety of the Disclosure would involve multiple administrations of a selected formulation over a period of several weeks or months. More specifically, the antibody or its antigen-binding moiety of the Disclosure may be administered once daily, every two days, every four days, once a week, every ten days, every two weeks, every three weeks, once a month, every six weeks, every two months, every ten weeks, or every three months. In this regard, it will be understood that the dosage may be changed or the intervals adjusted based on the patient's response and clinical practice.

[0158] Dosages and regimens may also be empirically determined for the therapeutic compositions of the Disclosure in individuals receiving one or more doses. For example, an individual may be given an increasing dose of the therapeutic composition prepared as described herein. In selected embodiments, doses may be gradually increased, decreased, or attenuated based on empirically determined or observed side effects or toxicity. To evaluate the efficacy of the selected composition, markers of specific diseases, disorders, or conditions may be tracked as described above. In the case of cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques, improvement as assessed by direct tumor biopsy and microscopic examination of tumor samples, measurement of indirect tumor markers (e.g., PSA in prostate cancer) or oncoplastic antigens identified according to the methods described herein, reduction of pain or paralysis, improvement of speech, vision, respiration, or other impairments related to the tumor, increased appetite, or improvement in quality of life as measured by approved tests, or extension of survival.

[0159] In some embodiments, suitable formulations for parenteral administration (e.g., intravenous injection) contain the antibody or its antigen-binding moiety 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 moiety disclosed herein varies depending on the individual, the type of neoplasm, the stage of the neoplasm, whether the neoplasm has begun to spread to other parts of the individual, past and concomitant treatments, and the dosage of any therapeutic agents used in combination with the antibody disclosed herein.

[0160] Uses of this disclosure The antibodies, antibody compositions, and methods of this disclosure have numerous in vitro and in vivo applications, including detection of TIGIT / PVRIG or enhancement of the immune response. For example, these molecules can be administered in vitro or ex vivo to cells in culture, or to human subjects, for example, in vivo, to enhance immunity in various situations. The immune response can be modulated, for example, enhanced, stimulated, or upregulated.

[0161] For example, the target population includes human patients who require enhancement of the immune response. This method is particularly suitable for treating human patients with disorders that can be treated by enhancing the immune response (e.g., T cell-mediated immune response). In certain embodiments, this method is particularly suitable for the in vivo treatment of cancer. When the bispecific antibody is administered together with another agent, such as an anti-PD-L1 agent, the two can be administered in any order or simultaneously.

[0162] Treatment of disorders, including cancer In some embodiments, the Disclosure provides a method for treating a mammalian disorder or disease, comprising administering a therapeutically effective amount of a bispecific antibody or its antigen-binding moiety disclosed herein to a subject in need of treatment (e.g., a human), in combination with, for example, a PD-1 / PD-L1 antagonist. The disorder or disease includes, but is not limited to, proliferative disorders (such as cancer), immune disorders, inflammatory diseases, or infections. For example, the disorder may be cancer.

[0163] In some embodiments, the cancer is characterized by high expression of CD112, CD113, or CD155. In some embodiments, the cancer is characterized by an abundance of TIGIT-expressing T cells or natural killer (NK) cells. In some embodiments, the cancer is characterized by high expression of PRVL2. In some embodiments, the cancer is characterized by an abundance of PVRIG-expressing T cells or natural killer (NK) cells.

[0164] Cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, 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) (NSCLC includes squamous cell NSCLC or non-squamous cell NSCLC, which include locally advanced unresectable NSCLC (e.g., stage IIIB NSCLC) or recurrent or metastatic NSCLC (e.g., stage IV) This includes lung adenocarcinoma or squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); esophageal cancer; peritoneal cancer; hepatocellular carcinoma; gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer (e.g., urothelial bladder cancer (UBC), muscle-invasive bladder cancer (MIBC), and BCG-resistant non-muscle-invasive bladder cancer (NMIBC)); urinary tract cancer; hepatocellular carcinoma; breast cancer (e.g., , TIGIT+ breast cancer and triple-negative breast cancer (TNBC) (estrogen receptor-negative (ER-), progesterone receptor-negative (PR-), and TIGIT-negative (TIGIT-)); colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; kidney cancer or renal cancer (e.g., renal cell carcinoma (RCC)); prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; superficial spreading melanin Melanomas, including chromoma, lentigo malignant melanoma, acral lentiginous melanoma, and nodular melanoma; multiple myeloma and B-cell lymphoma (low-grade / follicular non-Hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-incisional nuclear NHL; giant tumor NHL; mantle cell lymphoma; AIDS-associated lymphoma; Walde This includes Ström's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorders (PTLD); myelodysplastic syndromes (MDS); and abnormal angiogenesis associated with nevus syndromes, edema (such as that associated with brain tumors), Meigs syndrome, brain cancer, head and neck cancer, and associated metastases.

[0165] As co-inhibitory receptors on various immune cells, TIGIT or PVRIG are involved in various cancers, whether malignant or benign, primary or secondary, which can be treated or prevented in the manner provided by this disclosure. Cancers can be solid tumors or hematological malignancies. Examples of such cancers include lung cancers such as bronchogenic cancers (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondropathic hamartoma (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyoma; osteochondroma, chondroma, chondroblastoma, chondromyxofibroma, osteoid, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, and Ewing's tumor. Bone cancers such as Ewing's sarcoma and reticular sarcoma; gliomas (e.g., glioblastoma multiforme), undifferentiated astrocytoma, astrocytoma, oligodendroglioma, medulloblastoma, chordoma, schwannoma, ependymoma, meningioma, pituitary adenoma, pineal glandoma, osteoma, hemangioblastoma, craniopharyngioma, chordoma, germ cell tumor, teratoma, dermoid cystoma and hemangioma; colon cancer, leiomyoma, epidermoid carcinoma, adenocarcinoma, leiomyosarcoma, gastric adenocarcinoma, intestinal lipoma, intestinal neurofibroma, intestinal fibroma, colon polyps Cancers of the digestive system such as bladder and colorectal cancer; liver cancers such as hepatocellular adenoma, hemangioma, hepatocellular carcinoma, fibrolaminar carcinoma, cholangiocarcinoma, hepatoblastoma, and angiosarcoma; kidney cancers such as renal adenocarcinoma, renal cell carcinoma, hyperephroma, and transitional cell carcinoma of the renal pelvis; bladder cancer; skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease; head and neck cancers; eye-related cancers such as retinoblastoma and intraocular melanoma; benign angioplasty Examples include cancers of the male reproductive system such as prostate hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonic carcinoma, and choriocarcinoma); breast cancer; cancers of the female reproductive system such as uterine cancer (endometrial cancer), cervical cancer, ovarian cancer, vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancer (papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, or medullary carcinoma, etc.); pheochromocytoma (adrenal gland); non-cancerous proliferation of the parathyroid gland; and pancreatic cancer. In certain embodiments, the cancer is colon cancer.

[0166] In some other embodiments, the disorder or disease to be treated or prevented is an immune-related disorder. The immune-related disorder 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 anergy, or by reduced cytokine secretion, reduced proliferation, or reduced cytolytic activity. In some embodiments, T-cell dysfunction is characterized by T-cell exhaustion. In some embodiments, the T cells are CD4+ and CD8+ T cells. In some embodiments, the immune-related disorder is selected from the group consisting of unresolved acute infections, chronic infections, and reduced tumor immunity.

[0167] Stimulation of the immune response In some embodiments, the Disclosure further provides a method for enhancing (e.g., stimulating) an immune response in a subject, comprising administering an antibody of the Disclosure or an antigen-binding moiety thereof to the subject so as to enhance the immune response in the subject. For example, the subject is a mammal. In certain embodiments, the subject is a human.

[0168] The term "enhance the immune response," or its grammatical variations, means stimulating, inducing, increasing, improving, or enhancing any response of the mammalian immune system. The immune response can be a cellular response (i.e., cell-mediated, e.g., cytotoxic T lymphocyte-mediated) or a humoral response (i.e., antibody-mediated), and can be a primary or secondary immune response. An example of enhancing the immune response is CD4 +Enhancement of the immune response can be evaluated 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. Typically, the methods of the disclosure enhance the immune response in mammals compared to the immune response in untreated mammals or mammals not treated with the methods disclosed herein. In one embodiment, an antibody or its antigen-binding moiety is used to enhance the human immune response to a microbial pathogen (such as a virus). In another embodiment, an antibody or its antigen-binding moiety is used to enhance the human immune response to a vaccine. In one embodiment, the method enhances the cellular immune response, particularly the cytotoxic T cell response. 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. Antibodies or their antigen-binding portions may be used to enhance the human immune response to microbial pathogens (such as viruses) or vaccines.

[0169] Antibodies or their antigen-binding portions may be used alone as monotherapy, or in combination with chemotherapy, radiotherapy, targeted therapy, or cellular immunotherapy.

[0170] Combination with chemotherapy Antibodies or their antigen-binding portions may be used in combination with anticancer agents, cytotoxic agents, or chemotherapeutic agents.

[0171] The terms “anticancer agent” or “antiproliferative agent” mean any agent that can be used to treat cell proliferation disorders such as cancer, and include, but are not limited to, cytotoxic agents, cell proliferation inhibitors, anti-angiogenic agents, weight-reducing agents, chemotherapeutic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It will be understood that such anticancer agents may include conjugates and may be conjugated with the antibodies of this disclosure before administration. More specifically, in certain embodiments, an engineered conjugate is provided by linking a selected anticancer agent to an unpaired cysteine ​​of an engineered antibody. Thus, such an engineered conjugate is explicitly intended to be within the scope of this disclosure. In some other embodiments, the anticancer agent is given in combination with an antibody-drug conjugate containing a different therapeutic agent.

[0172] As used herein, the term “cytotoxic agent” means a substance that is toxic to cells, reduces or inhibits cellular function, and / or causes cell destruction. In certain embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-sarcin, restrictosin), plants (e.g., abrin, lysine, modesin, biscumin, pokeweed antiviral protein, saporin, geronin, momorizin, tricosanthin, barley toxin, Chinese tallow tree protein, dianthin protein, pokeweed protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, curcin, crotin, soapwort inhibitor, geronin, mitejerin, restrictosin, phenomycin, neomycin and trichothecene), or animals (e.g., cytotoxic RNases such as extracellular pancreatic RNase, DNase I (including its fragments and / or variants)).

[0173] For the purposes of this disclosure, “chemotherapeutic agents” include chemical compounds (e.g., cytotoxic agents or cell proliferation inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. Such chemical agents often target intracellular processes necessary for cell growth or division and are therefore generally particularly effective against cancer cells that grow and divide rapidly. For example, vincristine depolymerizes microtubules and, consequently, inhibits cells from entering mitosis. Generally, chemotherapeutic agents may include any chemical agents that inhibit or are designed to inhibit cancer cells or cells that are likely to become cancerous or produce tumorigenic offspring (e.g., TICs). Such agents are often administered and are most effective in combination with regimens such as CHOP or FOLFIRI.

[0174] Examples of anticancer agents that may be used in combination with the bispecific antibodies of this disclosure (either as components of a site-directed complex or in an unbound state) include alkylating agents, alkyl sulfonates, aziridines, ethyleneimines and methylamelamines, acetogenins, camptothecin, bryostatin, calistatin, CC-1065, cryptophycin, dorastatin, duocalmycin, eleuterobin, pancratistatin, sarcodicin, spongistin, nitrogen mustard, antibiotics, engine antibiotics, dinemycin, bis Phosphonate, Esperamycin, Pigment Protein Endiin Antibiotic Chromophore, Acrasinomycin, Actinomycin, Ausramycin, Azaserin, Bleomycin, Cactinomycin, Carabicin, Carminomycin, Cardinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-Oxo-L-Norleucine, ADRIAMYCIN®, Doxorubicin, Epirubicin, Esolubicin, Idarubicin, Marcelomycin, Mitomycin, Mycopheno Folic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, antimetabolites, erlotinib, vemurafenib, crizotinib, sorafenib, ibrutinib, enzalutamide, folic acid analogs, purine analogs, androgens, anti-adrenal agents, folic acid supplements such as folic acid, acegraton, aldofsphamide glycoside, aminolevulinic acid, ene Luuracil, Amsacrin, Bestrabusil, Bisanthren, Edatraxate, Dehofamine, Demecolsin, Diadicone, Elfornithine, Erliptinium acetate, Epotilon, Etoglucid, Gallium nitrate, Hydroxyurea, Lentinan, Ronidynin, Mytansinoid, Mitoguazone, Mitoxantrone, Mopidammol, Nitraerine, Pentostatin, Fenamet, Pirarubicin, Rosoxantrone, Podophyllic acid, 2-Ethylhydrazide, Procarbazine, PSK (Registered Trademark) Polysaccharide Complex (JHS Natural Products, Eugene, Oregon), Lazoxane, Rhizoxin, Schizophyllan, Spirogermanium, Tenuazonic acid, Triadicone, 2,2',2"-Trichlorotriethylamine, Trichothecenes (especially T-2 toxin, Beraclin A, Loridine A and Angidin), Urethane, Vindesine, Dacarbazine, Mannomustine, Mitobronitol, Mitractol, Pipobroman, Gacitosine, Arabinoside ("Ara-C"), Cyclophosphamide, Thiotepa, Taxoid, Chlorambucil, GEMZAR® Gemcitabine, 6-Thiogunine, Mercaptopurine, Methotrexate, Platinum Analogue, Vinblastine, Platinum, Etoposide (VP-16), Ifosfamide, Mitoxantrone, Vincristi Examples of pharmaceutically acceptable substances include, but are not limited to, navelbin (registered trademark), vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeroda, ibandronate, irinotecan (Camptosar, CPT-11), topoisomerase inhibitor RFS2000, difluoromethylornithine, retinoids, capecitabine, combretastatin, leucovorin, oxaliplatin, inhibitors of PKC-alpha, Raf, and VEGF-A that reduce cell proliferation, and any pharmaceutically acceptable salts, acids, or derivatives of the above. This definition also includes anti-hormone agents that act to control or inhibit hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, an enzyme that controls estrogen production in the adrenal gland, and anti-androgens, as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog), antisense oligonucleotides, ribozymes, such as VEGF expression inhibitors, vaccines, PROLEUKIN, (R) rIL-2, LURTOTECAN (R) Topoisomerase 1 inhibitor, ABARELIX (R) This also includes rmRH, vinorelbine, and esperamycin, as well as any pharmaceutically acceptable salt, acid, or derivative of any of the above.

[0175] Combined use with radiation therapy This disclosure also provides combinations of antibodies or their antigen-binding moieties with radiotherapy (i.e., any mechanism for inducing localized DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combination therapies using directed delivery of radioisotopes to tumor cells are also envisioned, and the antibodies of this disclosure may be used in conjunction with targeted anticancer agents or other targeting means. Typically, radiotherapy is administered in pulses over a period of about 1 to 2 weeks. Radiotherapy may be administered to subjects with head and neck cancer for about 6 to 7 weeks. Optionally, radiotherapy may be administered as a single dose or as a series of doses.

[0176] Pharmaceutical packs and kits Pharmaceutical packs and kits are also provided, comprising one or more containers containing one or more doses of antibody or its antigen-binding moiety. In certain embodiments, a unit dosage form is provided, comprising a predetermined amount of a composition containing, for example, an antibody or its antigen-binding moiety, together with or without one or more additional agents. In other embodiments, such a unit dosage form is supplied as a single-use pre-filled syringe for injection. In yet another embodiment, the composition contained in the unit dosage form may include, and / or be formulated within a stable and effective pH range, including, physiological saline, sucrose, and buffering agents such as phosphates. Alternatively, in certain embodiments, the composition may be provided as a lyophilized powder that is reconstituted upon addition of a suitable liquid, such as sterile water or physiological saline. In certain preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, for example, sucrose and arginine, but not limited to the following. Any label on or accompanying the container indicates that the encapsulated antibody is used to treat a selected neoplastic disease condition.

[0177] This disclosure also provides kits comprising single or multi-dose units of an antibody and, optionally, one or more anticancer agents. The kits comprise a container and labels or accompanying documents on or associated with the container. Suitable containers include, for example, bottles, vials, and syringes. Containers may be formed from a variety of materials, such as glass or plastic, and may contain a pharmaceutically effective amount of the antibody of this disclosure. In some embodiments, the container includes a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). Such kits generally comprise a pharmaceutically acceptable formulation of the antibody in a suitable container and, optionally, one or more anticancer agents in the same or different containers. The kits may also comprise other pharmaceutically acceptable formulations for either diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding moiety of the Disclosure, such kits may include one or more of various anticancer agents, such as chemotherapeutic agents or radiotherapy agents, anti-angiogenic agents, anti-metastatic agents, targeted anticancer agents, cytotoxic agents, and / or other anticancer agents. In some embodiments, the kit may include an anti-PD-1 antibody.

[0178] More specifically, the kit may have a single container containing the antibody or its antigen-binding moiety with or without additional components, or it may have separate containers for each desired drug. If concomitant therapeutic agents are provided for binding, a single solution may be pre-mixed in molar equivalent combinations or such that one component outweighs the other. Alternatively, the antibody and optional anticancer agents of the kit may be maintained separately in separate containers before administration to the patient. The kit may also include second / third container means for sterile, pharmaceutically acceptable buffers or other diluents, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and dextrose solution.

[0179] If the components of this kit are provided in one or more liquid solutions, the liquid solutions are preferably aqueous solutions, and sterile aqueous solutions or physiological saline are particularly preferred. However, the components of this kit may also be provided as dry powders. If the reagents or components are provided as dry powders, the powders can be reconstituted by adding a suitable solvent. It is also assumed that the solvent may be provided in a separate container.

[0180] As briefly stated above, the kit may also include means for administering the antibody or its antigen-binding portion and optional components to a patient, for example, one or more needles, IV bags or syringes, or eyedroppers, pipettes or other such instruments that can inject or introduce the formulation into an animal or apply it to a diseased area of ​​the body. The kit of the disclosure will also typically include means for tightly sealing and housing vials and other components for commercial sale, for example, injection-molded or blow-molded plastic containers that hold the desired vials or other instruments.

[0181] Sequence List Overview A sequence listing containing several amino acid sequences is attached to this application. Tables A, B, C, and D below provide an overview of the included sequences. The CDRs of the anti-TIGIT moiety or anti-TIGIT antibody are identified according to the Contact definition scheme, and the CDRs of the anti-PVRIG moiety or anti-PVRIG antibody are identified according to the IMGT / Kabat definition scheme (HCDR1 is defined by a combination of the IMGT and Kabat schemes, and other CDRs are defined by the Kabat scheme). W3XX104-T4U1.G15-2.uIgG1 and W3XX104-T4U1.G17-2.uIgG1 are bispecific antibodies constructed from the parent antibodies W3642 (anti-TIGIT) and WT1175 (anti-PVRIG).

[0182] [Table 0-1] [Table 0-2] [Table 0-3] [Table 0-4] [Examples]

[0183] The disclosure, broadly described above, will be more readily understood by referring to the following examples, which are provided as illustrations and are not intended to limit the disclosure. The examples are not intended to represent all or only experiments that have been conducted.

[0184] Example 1 Preparation of antigens, benchmark antibodies, and cell lines 1.1 Antigen Preparation W364-hPro1.ECD.His is the extracellular domain of human TIGIT (NP_776160.2) with a C-terminal polyhistidine tag, W364-hPro1.ECD.hFc is the extracellular domain of human TIGIT (NP_776160.2) with a human IgG1 Fc region at its C-terminus, and W364-mPro1.ECD.His is the extracellular domain of mouse T with a C-terminal polyhistidine tag. W364-mPro1.ECD.hFc is the extracellular domain of mouse TIGIT (NP_001139797.1), having the Fc region of human IgG1 at its C-terminus, while W364-hPro1L1.ECD.hFc is the extracellular domain of human CD155 (NP_006496.3), having the Fc region of human IgG1 at its C-terminus. W364-hPro1L1.ECD.mFc is the extracellular domain of human CD155 (NP_006496.3), having the Fc region of mouse IgG1 at its C-terminus.

[0185] WT117-hPro1.ECD.His is the extracellular domain of human PVRIG (NP_076975.2) with a C-terminal polyhistidine tag, WT117-hPro1.ECD.hFc is the extracellular domain of human PVRIG (NP_076975.2) with a human IgG1 Fc region at its C-terminus, and WT117-hPro1.ECD.mFc is the extracellular domain of human PVRIG (NP_076975.2) with a mouse IgG2a Fc region at its C-terminus. mPro1.ECD.His is the extracellular domain of mouse PVRIG (XP_011239268.1) with a C-terminal polyhistidine tag, WT117-mPro1.ECD.hFc is the extracellular domain of mouse PVRIG (XP_011239268.1) with a human IgG1 Fc region at its C-terminus, and WT117-hPro1L1.ECD.mFc is the extracellular domain of human PVRL2 (NP_001036189.1) with a mouse IgG2a Fc region at its C-terminus.

[0186] These antigens were either purchased from vendors or prepared in-house.

[0187] 1.2 Preparation of Benchmark Antibody (BMK) In the following experiments, anti-human TIGIT or PVRIG reference antibodies, referred to herein as WBP364-BMK1 and WBPT117-BMK1, were used as controls. The sequences of these two antibodies were synthesized according to the sequences disclosed in their respective patents, and this information is summarized in Table 1. The human IgG1 isotype control antibody is an isotype control.

[0188] [Table 1]

[0189] 1.3 Preparation of cell pools / cell lines The human TIGIT-expressing cell line W364-CHOK1.hPro1.2A11 was created using CHOK1 cells transfected with full-length human TIGIT (NP_776160.2). The cynomolgus monkey TIGIT-expressing cell pool W364-FlpinCHO.cynoPro1.pool was created using FlpinCHO cells transfected with full-length cynomolgus monkey TIGIT (XP_015300911.1). The mouse TIGIT-expressing cell pool W364-FlpinCHO.mPro1.pool was created using FlpinCHO cells transfected with full-length mouse TIGIT (NP_001139797.1).

[0190] The human PVRIG-expressing cell line WT117-293F.hPro1.G11 was created using 293F cells transfected with full-length human PVRIG (NP_076975.2). The cynomolgus monkey PVRIG-expressing cell pool WT117-Flpin293.cPro1.pool was created using Flpin293 cells transfected with full-length cynomolgus monkey PVRIG (XP_005549281.1). The mouse PVRIG-expressing cell pool WT117-293F.mPro1.pool was created using 293F cells transfected with full-length mouse PVRIG (XP_011239268.1).

[0191] Example 2 Production of bispecific antibodies 2.1 Plasmid Construction We developed monospecific anti-PVRIG antibodies and monospecific anti-TIGIT antibodies, respectively. The anti-TIGIT monoclonal antibody W3642 (or "T4") was obtained by immunizing and then humanizing Sprague-Dawley (SD) rats. The anti-PVRIG monoclonal antibody WT1175 (or "U1") was obtained by immunizing genetically modified OmniRat. The variable regions of these antibodies were extracted to construct bispecific anti-PVRIG / TIGIT antibodies.

[0192] Construction of G15 format bispecific antibody: The DNA sequence encoding the scFv (VH-(G4S)4-VL) of the anti-PVRIG antibody was ligated to the C-terminus of the full-length heavy chain of the anti-TIGIT antibody using a (G4S)4 linker (i.e., IgG(H)-scFv format). This sequence was cloned into the pcDNA3.4 expression vector. The resulting 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 in the left panel of Figure 1.

[0193] Construction of G17 format bispecific antibody: The DNA sequence encoding the scFV (VH-(G4S)4-VL) of the anti-TIGIT antibody was ligated to the N-terminus of the full-length heavy chain of the anti-PVRIG antibody using a (G4S)4 linker (i.e., scFv-IgG(H) format). This sequence was cloned into the pcDNA3.4 expression vector. The resulting 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 in the right panel of Figure 1.

[0194] The sequences of the above antibodies are summarized in Tables A - C above.

[0195] 2.2 Expression in Expi293 cells Expi293 cells (Thermo fisher, cat#A14635) were used for protein expression. Plasmids of the bispecific antibody heavy and light chains were transfected into Expi293 cells. After transfection, the cultures were incubated at 37 °C, 8% CO2 for 5 days, and then the supernatant was collected for protein purification.

[0196] 2.3 Antibody purification The supernatant of the Expi293 cell culture was collected, filtered, and purified using a Protein A column (GE Healthcare, cat#175438). The concentration of the purified antibody was measured by UV absorbance at 280 nm. The molecular weight and purity were tested by SDS-PAGE and SEC-HPLC, respectively.

[0197] Example 3 In vitro characterization 3.1 Human TIGIT or PVRIG binding assay W364-CHOK1.hPro1.2A11 cells or WT117-293F.hPro1.G11 cells were incubated with various concentrations of the antibody at 4°C for 1 hour. After washing with 1×PBS / 1% BSA, the secondary antibody, PE-labeled goat anti-human IgG (Jackson ImmunoReasearch, cat#109-115-098), was added and incubated with the cells at 4°C in the dark for 1 hour. The anti-human TIGIT antibody WBP364-BMK1 and the anti-PVRIG antibody WBPT117-BMK1 were used as positive controls, respectively. A human IgG1 isotype control antibody was used as an isotype control. Next, the cells were washed and resuspended in 1×PBS / 1% BSA. The MFI of the cells was measured by flow cytometry (BD) and analyzed with FlowJo.

[0198] The results of antibody binding to W364-CHOK1.hPro1.2A11 or WT117-293F.hPro1.G11 are shown in Figures 2 and 3. From these results, it can be seen that both G17 and G15 bind to human TIGIT-expressing cells W364-CHOK1.hPro1.2A11 with the same potency as WBP364-BMK1, and both G17 and G15 bind to PVRIG-expressing cells WT117-293F.hPro1.G11 with higher potency than WBPT117-BMK1. An overview of antibody binding is shown in Table 4.

[0199] 3.2 Cynomolgus TIGIT or PVRIG binding assay W364-FlpinCHO.cynoPro1.pool cells or WT117-Flpin293F.cPro1.pool cells were incubated with varying concentrations of antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, PE-labeled goat anti-human IgG (Jackson ImmunoResearch, cat#109-115-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-human TIGIT antibody WBP364-BMK1 and anti-PVRIG antibody WBPT117-BMK1 were used as positive controls, respectively. Human IgG1 isotype control antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular Filtration Intake (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0200] Figures 4 and 5 show the results of anti-TIGIT / PVRIG BsAb binding to W364-FlpinCHO.cynoPro1.pool or WT117-Flpin293F.cPro1.pool. These results show that both G17 and G15 bound to cynomolgus monkey TIGIT-expressing cells W364-FlpinCHO.cynoPro1.pool with the same potency as WBP364-BMK1, and that both G17 and G15 bound to PVRIG-expressing cells WT117-Flpin293F.cPro1.pool with higher potency than WBPT117-BMK1. A summary of antibody binding is shown in Table 4.

[0201] 3.3 Mouse TIGIT binding assay W364-FlpinCHO.mPro1.pool cells were incubated with various concentrations of antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, PE-labeled goat anti-human IgG (Jackson ImmunoResearch, cat#109-115-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Human IgG1 isotype control antibody was used as an isotype control. The cells were then washed and resuspended in 1×PBS / 1%BSA. Cellular Filtration Intake (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0202] Figure 6 shows the results of anti-TIGIT / PVRIG BsAb binding to W364-FlpinCHO.mPro1.pool cells. These results show that both G17 and G15 bind to mouse TIGIT-expressing cells, while WBP364-BMK1 does not. Table 4 summarizes the antibody binding process.

[0203] 3.4 Mouse PVRIG binding assay WT117-293F.mPro1.pool cells were incubated with various concentrations of antibody at 4°C for 1 hour. After washing with 1×PBS / 1%BSA, the secondary antibody, PE-labeled goat anti-human IgG (Jackson ImmunoResearch, cat#109-115-098), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-mouse PVRIG antibody fused to 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 1×PBS / 1%BSA. Cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0204] Figure 7 shows the binding results of anti-TIGIT / PVRIG BsAb to WT117-293F.mPro1.pool cells. These results indicate that neither G17, G15, nor WBPT117-BMK1 bind to mouse PVRIG.

[0205] 3.5 Human TIGIT or PVRIG Binding Affinity Assay The affinity of anti-TIGIT / PVRIG BsAbs for recombinant human TIGIT or PVRIG was measured by surface plasmon resonance (SPR) using a Biacore 8K instrument (Cytiva). Goat anti-human IgG Fc antibody (Jackson ImmunoResearch, cat#109-005-098) was immobilized on a CM5 biosensor chip (GE, cat#29-1496-03), and the test antibody was captured by the goat anti-human IgG Fc antibody. For reaction kinetics, a series of concentrations of W364-hPro1.ECD.His or WT1175-hPro1.ECD.His were injected into running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% surfactant P20, pH 7.4) at a flow rate of 30 μL / min at 25°C for the binding phase, followed by a dissociation phase for a certain period. 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.

[0206] Tables 2 and 3 show the SPR binding affinity results of anti-TIGIT / PVRIG BsAbs to human TIGIT or PVRIG. G17 binds to human TIGIT and PVRIG proteins with affinities of 1.17E-11M and 2.69E-09M, respectively. G15 binds to human TIGIT and PVRIG proteins with affinities of 1.87E-11M and 5.42E-09M, respectively. A summary of antibody binding affinity is shown in Table 4, and sensorgrams are shown in Figures 8 and 9.

[0207] [Table 2]

[0208] [Table 3]

[0209] 3.6 Paralog protein binding assay Plates were 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 1×PBS / 2%BSA, 100 μL of test antibody at a concentration of 66.67 nM was added to the plate and incubated at room temperature for 1 hour. After incubation, the plate was washed three times with 1×PBST. HRP-labeled goat anti-human IgG antibody (Bethyl, cat#A80-304P) diluted in 1×PBS / 2%BSA was added and incubated at room temperature for 1 hour. After washing six times with 1×PBST, 100 μL of TMB substrate was dispensed and allowed to develop color, then 100 μL of 2M HCl was added to stop the reaction. Absorbance at 450 nm and 540 nm was read using an M5e microplate reader (Molecule Devices).

[0210] Figure 10 shows the binding results of anti-TIGIT / PVRIG BsAb to the paralog proteins of TIGIT and PVRIG. These results indicate that both G17 and G15 specifically bind to TIGIT and PVRIG without cross-reactivity to human CD226, CD96, and PD-1.

[0211] 3.7 Human TIGIT and PVRIG double bond assay Plates were pre-coated overnight at 4 °C with 0.1 μg / mL of W364-hPro1.ECD.His in 100 μL of coating buffer per well. After blocking with 200 μL of 1×PBS / 2% BSA, 100 μL of test antibody was added to the plates at various concentrations and incubated for 2 hours at room temperature. After incubation, the plates were washed three times with 1×PBST. WT117-hPro1.ECD.mFc was diluted to a concentration of 0.1 μg / mL in 1×PBS / 2% BSA and added to the plates. After incubation for 1 hour at room temperature, the plates were washed three times with 1×PBST. HRP-labeled goat anti-mouse IgG antibody (Bethyl, cat#A90-231P) diluted in 1×PBS / 2% BSA was added and incubated for 1 hour at room temperature. After washing six times with 1×PBST, 100 μL of TMB substrate was dispensed to develop color, and then 100 μL of 2 M HCl was added to stop the reaction. Absorbance at 450 nm and 540 nm was read using an M5e microplate reader (Molecule Devices).

[0212] Results of the dual binding of anti-TIGIT / PVRIG BsAb to immobilized human TIGIT and soluble human PVRIG are shown in Fig. 11. From these results, it can be seen that both G17 and G15 bind simultaneously to human TIGIT and PVRIG, while the reference mAbs, WBP364-BMK1 and WBPT117-BMK1, do not dual-bind to TIGIT and PVRIG. An overview of antibody dual binding is shown in Table 4.

[0213] 3.8 Human TIGIT / PVR Blocking Assay The ability of anti-TIGIT / PVRIG BsAb to block the human TIGIT / PVR interaction was tested by FACS. W364-CHOK1.hPro1.2A11 cells were washed with 1×PBS / 1% BSA and 1×10 per well were added to a 96-well round-bottom plate 5The cells were seeded to form individual cells. Excess buffer in the wells was removed by centrifugation. Serially diluted antibodies (2× concentration) and 4 μg / mL (2× concentration) W364-hPro1L1.ECD.mFc were pre-mixed in 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 1× PBS / 1% BSA, the secondary antibody, PE-labeled goat anti-mouse IgG (Bethyl, cat#A90-239PE), was added, and the cells were incubated with the antibody at 4°C in the dark for 1 hour. Anti-human TIGIT antibody WBP364-BMK1 was used as a positive control. Human IgG1 isotype antibody was used as an isotype control. The cells were then washed and resuspended in 1× PBS / 1% BSA. Cellular cellular filtration efficiency (MFI) was measured by flow cytometry (BD) and analyzed by FlowJo.

[0214] The results of blocking human TIGIT / PVR are shown in Figure 12. These results show that both G17 and G15 blocked the binding of human PVR to human TIGIT, demonstrating blocking efficacy equivalent to that of the reference TIGIT mAb, WBP364-BMK1. A summary of antibody blocking activity is shown in Table 4. The maximum inhibition rate is given by: Inhibition Rate % = (MFI max -MFI bottom ) / MFI max It was calculated using a multiplier of 100%. Here, MFI max This was defined as MFI in the absence of antibodies.

[0215] 3.9 Human PVRIG / PVRL2 Blocking Assay 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 1× PBS / 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 room temperature for 1 hour. Serially diluted antibodies (2× concentration) were mixed with 0.6 μg / mL (2× concentration) of WT1175-hPro1L1.ECD.mFc in a 1:1 volume ratio. After incubation, 100 μL of the antibody / ligand mixture was added to the plate and incubated at room temperature for 2 hours. After washing three times with 1×PBST, HRP-labeled goat anti-mouse IgG (Bethyl, cat#A90-231P) was added to the plate and incubated at room temperature for 1 hour. After washing six times with 1×PBST, TMB substrate was added, and the interaction was stopped with 2M HCl. Absorbance at 450 nm and 540 nm was read using an M5e microplate reader (Molecule Devices).

[0216] Figure 13 shows the results of blocking human PVRIG / PVRL2. These results indicate that both G17 and G15 blocked the binding of human PVRL2 to human PVRIG, demonstrating blocking efficacy equivalent to that of the reference PVRIG mAb, WBPT117-BMK1. Table 4 summarizes the antibody blocking activity. The maximum inhibition rate is given by: Inhibition Rate % = (OD max -OD bottom ) / OD max It was calculated as ×100%. Here, OD max This was defined as the (OD450-OD540) value in the absence of the antibody.

[0217] 3.10 Jurkat TIGIT / PVRIG / NFAT-luciferase reporter gene assay (RGA) Jurkat cells overexpressing human TIGIT, PVRIG, and NFAT luciferase reporters were stimulated by co-culturing them with artificial APCs to conjugate T cell receptors. The artificial APCs were engineered HT1080 cells constitutively expressing PVR and PVRL2, and were transfected to express human TCR activators. HT1080 / TCR activator cells were cultured in 96-well plates at a rate of 2 × 10⁶ cells. 4 The cells were seeded at a density of cells / well and left overnight at 37°C in 5% CO2. On the second day, after removing the supernatant and non-adherent cells, serially diluted antibodies were added to Jurkat / TIGIT / PVRIG / NFAT-luciferase cells (4 × 10⁶). 4 The cells (per well) were added to the plate and co-cultured at 37°C and 5% CO2 for 5.5 hours. After incubation, the reconstituted luciferase substrate (Promega, cat#E605B) was added to each well and mixed thoroughly. Luciferase intensity was read using an Envision microplate reader (PerkinElmer).

[0218] The results of the Jurkat TIGIT / PVRIG / NFAT-luciferase reporter gene assay are shown in Figure 14. These results show that both G17 and G15 dramatically enhanced TCR / NFAT signaling by blocking the interaction between TIGIT / PVR and PVRIG / PVRL2. Importantly, the potency and effectiveness of the BsAb were higher than those observed when using the reference mAbs WBP364-BMK1 or WBPT117-BMK1 alone or in combination. Table 4 summarizes the antibody activity that reversed TCR / NFAT suppression. The ratio of change was defined as the ratio of the luciferase value in the sample to the luciferase value in the absence of the antibody.

[0219] 3.11 Human Primary NK Cell Killing Assay HT1080 is a human fibrosarcoma cell line that expresses human PVR, PVRL2, and PVRL3. HT1080 expressing human PVR and PVRL2 was used as the target cell, and human primary NK cells were used as effector cells. Human primary NK cells were isolated from human peripheral blood mononuclear cells (PBMCs) by magnetic selection using human CD56 MicroBeads (Miltenyi Biotec, cat#130-050-401) according to the manufacturer's protocol. The isolated human NK cells (4 × 10⁶) 4 HT1080 cells (2 × 10¹⁶ cells / well) were placed in the presence of serially diluted antibodies. 4 The cells were co-cultured with the target cells (individual cells / well) at 37°C and 5% CO2 for 8 hours. Lysis of the target cells was measured using an LDH-based cytotoxicity detection kit (Roche, cat#04744934001). Absorbance at 492 nm was read using an M5e microplate reader (Molecule Devices).

[0220] The results of the NK cell killing assay are shown in Figure 15. These results show that both G17 and G15 dose-dependently enhanced the cytotoxicity of primary NK cells, and their efficacy was higher than that of WBP364-BMK1 and WBPT117-BMK1 used individually or in combination. Table 4 outlines the antibodies that enhance NK cell activity.

[0221] 3.12 Human Primary T Cell Activation Assay Human first CD8 + T cells were stimulated by co-culturing them with artificial APCs to conjugate T cell receptors. The artificial APCs were engineered HT1080 cells constitutively expressing PVR and PVRL2, and were transfected to express human TCR activator. Human CD8 + T cells were isolated from human PBMCs using magnetic selection with human CD8 MicroBeads (Miltenyi Biotec, cat#130-045-201) according to the manufacturer's protocol. +T cells were co-cultured with human T activator CD3 / CD28 Dynabeads (Gibco, cat#11132D) for 7 days to induce exhaustion. After removing the magnetic beads with a magnet, these exhausted T cells (1 × 10⁶ per well) were extracted. 5 HT1080 / TCR activator cells (1 x 10 cells per well) treated with mitomycin C in the presence of serially diluted antibodies. 4 The cells were co-cultured with [specimen number]. After incubation for 5 days, the supernatant was collected and IFN-γ was measured by ELISA (capture antibody Thermo cat#M700A, detection antibody Thermo cat#M701B). Absorbance was detected using an M5e microplate reader (Molecule Devices).

[0222] The results of the T cell activation assay are shown in Figure 16. From these results, both G17 and G15 are exhausted human CD8 cells. + The antibodies enhance T cell activation in a dose-dependent manner, and their efficacy is higher than that of WBP364-BMK1 and WBPT117-BMK1 used alone or in combination. Table 4 shows an overview of the antibodies that enhance T cell activity.

[0223] [Table 4]

[0224] 3.13 Feasibility of antibody development (1) Thermal stability assay Conformational stability is a crucial property for promising antibody candidates. Conformational stability can be evaluated by measuring thermal stability using differential scanning fluorescence (DSF), which can detect changes in protein folding. DSF measures the unfolding transition temperature (Tm) of a protein based on changes in the fluorescence intensity of the environmentally sensitive dye SYPRO Orange.

[0225] DSF was performed using a Quant Studio 7 Flex real-time PCR instrument (Applied Biosystems) in each formulation buffer. SYPRO Orange dye (Invitrogen, cat#S6651) was added to the antibody, and the mixture was transferred to a 96-well plate. The plate was then transferred to a Quant Studio® 7 Flex real-time PCR instrument with a temperature range of 26°C to 95°C and a heating rate of 0.9°C / min. The first two temperatures during protein unfolding were recorded as Tm1 and Tm2. These two values ​​were calculated according to the melting curve using Quant Studio® real-time PCR software (v1.3).

[0226] The DSF results are shown in Table 5 and Figure 17. The DSF thermogram of BsAb shows the following two transitions, where Tm1 reflects the dissolution of the CH2 domain and Tm2 reflects the melting temperatures of CH3 and Fab. The Tm1 for G17 and G15 were 57.1°C and 55.6°C, respectively, and the Tm2 were 64.7°C and 63.6°C, respectively. From these results, it can be seen that both G17 and G15 have good thermal stability.

[0227] [Table 5]

[0228] (2) Solubility test using DLS-kD Dynamic light scattering (DLS) is widely used to evaluate protein aggregation and stability. The diffusion interaction parameter (kD) indicates how the diffusion rate of a protein is affected by protein concentration as a result of intermolecular forces.

[0229] DLS was performed using a DynaPro Plate Reader III (Wyatt Technology) to determine the kD value of the antibodies. Antibody concentration series (20, 15, 10, 5, and 2.5 mg / mL) were prepared, and 7.5 μL of sample dilution was added to a 1536-well microplate. The plate was sealed with ClearSeal Film (Hampton Research, cat#HR4-521), and aggregates were removed by centrifugation at 3000 rpm for 5 minutes. Each sample was tested twice. The 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 the sample concentration. The slope of the plotted line represents the kD value of the sample.

[0230] The DLS-kD results are shown in Table 6 and Figure 18. The kD values ​​for G17 and G15 were -7.29 mL / g and -7.24 mL / g, respectively. No aggregation or particles were observed during the experiment. These results indicate that both G17 and G15 have good solubility.

[0231] [Table 6]

[0232] (3) Hydrophobicity test by HIC-HPLC Hydrophobic interaction chromatography—high-performance liquid chromatography (HIC-HPLC)—is a powerful analytical method used to predict the relative hydrophobicity of therapeutic proteins. The retention time of the protein in question reflects the overall hydrophobicity of the protein.

[0233] To predict the hydrophobicity of the antibody, retention times were calculated using an HPLC1260 Infinity II system (Agilent Technologics®) equipped with a TSKgel butyl-NPR column (Tosoh, cat#0042168). Each sample was diluted to 0.5 mg / mL in PBS, 20 μL was injected into the column, and the separation flow rate was set to 0.5 mL / min for 61 minutes. The running buffer was 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 using a gradient of running buffer (buffer D from 0% to 100%) for 3 to 53 minutes. Peak retention times were measured by detecting UV absorbance at 280 nm and 230 nm. The retention time was calculated by integrating all peak areas from 20 minutes to 40 minutes using OpenLab CDS Workstation (v2.6.0.691) software.

[0234] The HIC-HPLC results are shown in Table 7 and Figure 19. The retention times for G17 and G15 were 30.31 minutes and 28.62 minutes, respectively, which reflect normal hydrophobicity.

[0235] [Table 7]

[0236] (4) Stress tests at 4°C and 40°C 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% PS80, 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, respectively, and stored in a freezer set to -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 changes in high molecular weight (HMW), monomer (Mono), and low molecular weight (LMW) species present in the solution were analyzed by SEC-HPLC.

[0237] The results are shown in Tables 8 and 9. Under thermal stress at 40°C, G17 showed a slight increase in LMW proportion and a slight decrease in Mono proportion. In G15, GMW proportion increased slightly and Mono proportion decreased slightly. No significant changes were observed in protein concentration.

[0238] [Table 8]

[0239] [Table 9]

[0240] (5) Stress test after freeze-thaw cycle 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 room temperature, refreezed at -80°C, and thawed again. After a total of five F / T cycles, samples were taken, their physical appearance was evaluated by observation, their concentration was determined by UV absorbance at 280 nm, and changes in HMW, Mono, and LMW species present in the solution were evaluated by SEC-HPLC.

[0241] The results are shown in Tables 10 and 11. After five F / T cycles, no effect was observed on the stability, appearance, concentration, or relative proportions of HMW, Mono, and LWM species of G17 and G15.

[0242] [Table 10]

[0243] [Table 11]

[0244] Example 4 In vivo characteristic evaluation 4.1 A 4-week pilot pharmacokinetic study in cynomolgus monkeys The objective of this study was to evaluate the pharmacokinetic profile of anti-TIGIT / PVRIG BsAb after repeated intravenous infusion in untreated male and female cynomolgus monkeys.

[0245] This study used eight healthy crab-eating macaques (four males and four females). Basic information is shown in Table 12.

[0246] [Table 12]

[0247] The bispecific antibody W3XX104-T4U1.G15-2.uIgG1 or W3XX104-T4U1.G17-2.uIgG1 was administered intravenously to one male and one female cynomolgus monkey at doses of 50 mg / kg / dose or 125 mg / kg / dose. The drug regimen consisted of four weekly administrations (IV infusion on days 0, 7, 14, and 21). This was a non-terminal study, and no scheduled necropsy was planned. The animals were returned to a relaxation room for washout and rest. The study endpoints included routine observation, body weight, serum cytokine concentration, and pharmacokinetics.

[0248] Animal body weight was recorded twice a week. Serum samples were collected at indicated time points (after initial administration, -7 days, -2 days, day 0 (1 hour and 8 hours after administration), day 1, day 2, day 3, day 5, day 7 (before administration), day 11, day 14 (before administration), day 18, day 21 (before administration, 1 hour and 8 hours after administration), day 22, day 23, day 24, day 26, and day 28) for pharmacokinetic analysis. After centrifugation, serum was collected and stored at -80°C until sent for analysis. The concentration of test BsAb in monkey serum was analyzed by ELISA. Briefly, goat anti-human IgG (Southern Biotech, cat#2049-01) was used as the capture reagent and biotinylated goat anti-human IgG (Southern Biotech, cat#2049-08) was used as the detection reagent. HRP-labeled streptavidin (Thermo, cat#SNN1004) and TMB substrate were used to develop a color, and the reaction was stopped with 2M HCl. Absorbance at 450nm and 540nm was read using an M5e microplate reader (Molecule Devices). Serum concentrations of test BsAb were determined from a 4-parameter fit of a standard curve. Concentrations were subjected to non-compartment pharmacokinetic analysis using Phoenix WinNonlin software (version 8.1, Pharsight). A linear / logarithmic trapezoidal rule was applied to the calculation of PK parameters.

[0249] Intravenous administration of W3XX104-T4U1.G17-2.uIgG1 and W3XX104-T4U1.G15-2.uIgG1 at doses of 50 mg / kg and 125 mg / kg once weekly for 4 weeks was well tolerated in cynomolgus monkeys, and all animals survived until the end of the study.

[0250] No significant changes in body weight were observed during the study with the bispecific antibodies T4U1.G17 and T4U1.G15 (Figure 20).

[0251] Exposure was confirmed in all animals by measuring serum PK. PK profiles are shown in Figures 21-24, and PK parameters are summarized in Table 13. For T4U1.G17, the mean Cmax values ​​were 1890 μg / mL at 50 mg / kg and 4980 μg / mL at 125 mg / kg, with the mean AUC being the highest. 0-t The values ​​were 155,645 h*μg / mL for 50 mg / kg and 539,834 h*μg / mL for 125 mg / kg. For T4U1.G15, the mean Cmax value was 1335 μg / mL for 50 mg / kg and 3305 μg / mL for 125 mg / kg, with the mean AUC being the highest. 0-t The values ​​were 266,105 h*μg / mL at 50 mg / kg and 945,971 h*μg / mL at 125 mg / kg. 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 0-t The values ​​increased more than proportionally to the dose. Based on serum concentrations after four administrations (days 1-28), the mean half-lives of T4U1.G17 were 54.5 hours and 56.3 hours, respectively, in the 50 mg / kg and 125 mg / kg groups, and the mean half-lives of T4U1.G15 were 63.5 hours and 72.5 hours, respectively. Based on serum concentrations after one administration (days 1-7), the mean half-lives of T4U1.G17 were 55.1 hours and 73.9 hours, respectively, in the 50 mg / kg and 125 mg / kg groups, and the mean half-lives of T4U1.G15 were 90.8 hours and 94.5 hours, respectively. No sex differences were observed in the 50 mg / kg and 125 mg / kg test doses.

[0252] [Table 13]

[0253] 4.2 In vivo efficacy of anti-TIGIT / PVRIG BsAb in a CT26 colon cancer cell model This study was conducted to evaluate the in vivo efficacy of the test substance in a syngeneic CT26 colon cancer cell model using human TIGIT and PVRIG transgenic BALB / c mice (BALB / c-hTIGIT / hPVRIG mice). CT26 cells (1 × 10⁶) 6 The drug (100 μL / mouse) was subcutaneously inoculated into 6-8 week old BALB / c-hTIGIT / hPVRIG mice (supplied by GemPharmatech). The average tumor volume was 65.85 mm². 3 Upon reaching a certain level, 40 tumor-bearing mice were randomly divided into five groups (n=8 / group) according to tumor volume. Mice in group G1 were administered PBS. Mice in groups G2-G4 were administered 1.3mpk, 4mpk, and 13.2mpk of W3XX104-T4U1.G15-2.uIgG1, respectively. Mice in group G5 were administered 10mpk of WBPT117-BMK1 (COM701) and 10mpk of the anti-TIGIT mAb WBP364-BMK1 (tiragolumab). COM701 is manufactured according to the sequence of U.S. Patent Application Publication No. 2019 / 0382477 (Table 1, clone ID: CHA.7.518.1). Tiragolumab is manufactured according to the sequence of INN PROP.LIST 117.

[0254] All mice received intraperitoneal administration (ip) twice a week (BIW) for a total of five times. Tumor volume and body weight were measured twice a week (days 0, 4, 7, 11, 14, and 17). Tumor volume was calculated using the formula: TV = 0.5a × b 2 Using mm 3 This is expressed as follows: In the formula, a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition rate (TGI) based on tumor volume. TV ) and tumor growth inhibition rate (TGI) based on tumor weight TW The in vivo efficacy of the test substance was evaluated by measuring ( ). Data are expressed as mean ± standard error (mean ± SEM). An independent sample t-test was performed to compare the two groups. P < 0.05 was considered statistically significant. GraphPad Prism9 was used for data visualization.

[0255] [Table 14]

[0256] Figure 28 shows the changes in body weight in each group. No significant weight loss was observed in any group. Figures 25, 26, and Table 15 show the changes in tumor volume in each group of mice. Figure 27 shows the changes in tumor weight in each group of mice. Table 16 shows the statistical analysis and p-values ​​for tumor growth inhibition in each group.

[0257] [Table 15]

[0258] Based on statistical analysis of tumor volume at the end of treatment (day 17), W3XX104-T4U1.G15-2.uIgG1 showed dose-dependent tumor growth inhibition at doses of 1.3 mg / kg, 4 mg / kg, and 13.2 mg / kg, indicating TGI TV The rates were 62.08%, 76.75%, and 86.69%, respectively (W3XX104-T4U1.G15-2.uIgG1 treatment group vs. vehicle control, P<0.05). Furthermore, tumor suppression in the W3XX104-T4U1.G15-2.uIgG1 monotherapy group (13.2 mg / kg) was comparable to that in the COM701 and tiragolumab combination group (10+10 mg / kg) (TGI: 87.30% vs. 86.69%, P>0.05).

[0259] [Table 16]

[0260] Based on statistical analysis of tumor weight at the end of treatment (day 17), W3XX104-T4U1.G15-2.uIgG1 showed dose-dependent tumor growth inhibition at doses of 1.3 mg / kg, 4 mg / kg, and 13.2 mg / kg, indicating TGI TWThe rates were 65.29%, 77.44%, and 89.04%, respectively (W3XX104-T4U1.G15-2.uIgG1 treatment group vs. vehicle control, P<0.05). Furthermore, tumor suppression in the W3XX104-T4U1.G15-2.uIgG1 monotherapy group (13.2 mg / kg) was comparable to that in the COM701 and tiragolumab combination group (10+10 mg / kg) (TGI). TW (89.04% vs. 88.05%, P>0.05).

[0261] In summary, this study demonstrated that W3XX104-T4U1.G15-2.uIgG1 could inhibit tumor volume (P<0.01) and tumor weight (P<0.001) in a dose-dependent manner (1.3 mpk, 4 mpk, and 13.2 mpk) compared to the vehicle control. Furthermore, monotherapy with W3XX104-T4U1.G15-2.uIgG1 (G4, 13.2 mpk of W3XX104-T4U1.G15-2.uIgG1, equivalent in molecular weight to COM701 or 10 mpk of tiragolumab) could produce a comparable tumor suppressive effect to combination therapy with COM701 and tiragolumab (G5, COM701 + tiragolumab, 10 mpk + 10 mpk).

[0262] Those skilled in the art will further understand that this disclosure can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of this disclosure discloses only exemplary embodiments, it should be understood that other modifications are also within the scope of this disclosure. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, the appended claims should be used to illustrate the scope and content of the invention.

[0263] References [1]Manieri NA, Chiang EY, Grogan JL.TIGIT:A Key Inhibitor of the Cancer Immunity Cycle.Trends Immunol.2017 Jan;38(1):20-28. [2]Rotte A,Jin JY,Lemaire V.Mechanistic overview of immune checkpoints to support the rational design of their combinations in cancer immunotherapy.Ann Oncol.2018 Jan 1;29(1):71-83. [3]Yu X,Harden K,et al.The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells.Nat Immunol.2009 Jan;10(1):48-57. [4]Joller N,et al.Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses.Immunity.2014 Apr 17;40(4):569-81. [5]Stanietsky N,et al.The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity.Proc Natl Acad Sci U S A.2009 Oct 20;106(42):17858-63. [6]Stengel KF,et al.Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering.Proc Natl Acad Sci USA.2012 Apr 3;109(14):5399-404. [7]Stanietsky N,et al.Mouse TIGIT inhibits NK-cell cytotoxicity upon interaction with PVR.Eur J Immunol.2013 Aug;43(8):2138-50. [8]Reches A,et al.Nectin4 is a novel TIGIT ligand which combines checkpoint inhibition and tumor specificity.J Immunother Cancer.2020 Jun;8(1):e000266. [9]Lozano E,Dominguez-Villar M,Kuchroo V,Hafler DA.The TIGIT / CD226 axis regulates human T cell function.J Immunol.2012 Apr 15;188(8):3869-75.

[10] Pauken KE,Wherry EJ.TIGIT and CD226:tipping the balance between costimulatory and coinhibitory molecules to augment the cancer immunotherapy toolkit.Cancer Cell.2014 Dec 8;26(6):785-787.

[11] Stamm H,Wellbrock J,Fiedler W.Interaction of PVR / PVRL2 with TIGIT / DNAM-1 as a novel immune checkpoint axis and therapeutic target in cancer.Mamm Genome.2018 Dec;29(11-12):694-702.

[12] Chauvin JM,et al.TIGIT and PD-1 impair tumor antigen-specific CD8 +T cells in melanoma patients.J Clin Invest.2015 May;125(5):2046-58.

[13] Kong Y,et al.T-Cell Immunoglobulin and ITIM Domain (TIGIT) Associates with CD8+ T-Cell Exhaustion and Poor Clinical Outcome in AML Patients.Clin Cancer Res.2016 Jun 15;22(12):3057-66.

[14] Johnston RJ,et al.The immunoreceptor TIGIT regulates antitumor and antiviral CD8(+) T cell effector function.Cancer Cell.2014 Dec 8;26(6):923-937.

[15] Fleury M,et al.Increased Expression and Modulated Regulatory Activity of Coinhibitory Receptors PD-1,TIGIT,and TIM-3 in Lymphocytes From Patients With Systemic Sclerosis.Arthritis Rheumatol.2018 Apr;70(4):566-577.

[16] Fromentin R,et al.CD4+ T Cells Expressing PD-1,TIGIT and LAG-3 Contribute to HIV Persistence during ART.PLoS Pathog.2016 Jul 14;12(7):e1005761.

[17] Galletti G,et al.Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans.Nat Immunol.2020 Dec;21(12):1552-1562.

[18] Banta KL,et al.Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.Immunity.2022 Mar 8;55(3):512-526.e9.

[19] Zhu Y,et al.Identification of CD112R as a novel checkpoint for human T cells.J Exp Med.2016 Feb 8;213(2):167-76.

[20] Xu F,Sunderland A,Zhou Y,Schulick RD,Edil BH,Zhu Y.Blockade of CD112R and TIGIT signaling sensitizes human natural killer cell functions.Cancer Immunol Immunother.2017 Oct;66(10):1367-1375.

[21] Murter B,et al.Mouse PVRIG Has CD8+ T Cell-Specific Coinhibitory Functions and Dampens Antitumor Immunity.Cancer Immunol Res.2019 Feb;7(2):244-256.

[22] Whelan S,et al.PVRIG and PVRL2 Are Induced in Cancer and Inhibit CD8+ T-cell Function.Cancer Immunol Res.2019 Feb;7(2):257-268.

[23] Sanchez-Correa B,et al.DNAM-1 and the TIGIT / PVRIG / TACTILE Axis:Novel Immune Checkpoints for Natural Killer Cell-Based Cancer Immunotherapy.Cancers (Basel).2019 Jun 23;11(6):8

[24] Zhang Z,Wu N,Lu Y,Davidson D,Colonna M,Veillette A.DNAM-1 controls NK cell activation via an ITT-like motif.J Exp Med.2015 Nov 16;212(12):2165-82.

[25] Alteber Z,Kotturi MF,Whelan S,Ganguly S,Weyl E,Pardoll DM,Hunter J,Ophir E.Therapeutic Targeting of Checkpoint Receptors within the DNAM1 Axis.Cancer Discov.2021 May;11(5):1040-1051.

Claims

1. A polypeptide complex comprising a TIGIT binding site and a PVRIG binding site, The TIGIT bond portion includes a heavy chain variable region (VH) containing heavy chain CDRs (HCDRs) 1, HCDRs 2 and HCDRs 3, and a light chain variable region (VL) containing light chain CDRs (LCDRs) 1, LCDRs 2 and LCDRs 3. The above HCDR1 includes the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 1; the above HCDR2 includes the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 2; the above HCDR3 includes the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 3; the above LCDR1 includes the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 4; the above LCDR2 includes the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 5; the above LCDR3 includes the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 6; A polypeptide complex in which the above substitutions are conservative substitutions.

2. The above PVRIG binding portion includes VH containing HCDR1, HCDR2, and HCDR3, and VL containing LCDR1, LCDR2, and LCDR3. The polypeptide complex according to claim 1, wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 7; HCDR2 comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 8; HCDR3 comprises the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 9; LCDR1 comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 10; LCDR2 comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 11; and LCDR3 comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having one, two, or three or fewer amino acid substitutions compared to SEQ ID NO: 12, wherein the substitutions are conservative substitutions.

3. The polypeptide complex according to claim 1 or 2, wherein the TIGIT binding portion and the PVRIG binding portion are either in Fab format or scFv format.

4. The polypeptide complex according to claim 1 or 2, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the TIGIT binding portion each contain the amino acid sequences of SEQ ID NOs: 1 to 6, and HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the PVRIG binding portion each contain the amino acid sequences of SEQ ID NOs: 7 to 12.

5. The VH of the TIGIT binding portion includes the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 13, and / or The polypeptide complex according to any one of claims 1 to 4, wherein the VL of the TIGIT binding portion includes the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:

14.

6. The VH of the PVRIG binding portion includes the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO: 15, and / or The polypeptide complex according to any one of claims 1 to 5, wherein the VL of the PVRIG binding portion includes the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 85%, 90%, or 95% identity with SEQ ID NO:

16.

7. The above TIGIT junction is in Fab format, and the above PVRIG junction is in scFv format, The above TIGIT junction is in scFv format, and the above PVRIG junction is in Fab format, or The polypeptide complex according to any one of claims 1 to 6, wherein both the TIGIT binding portion and the PVRIG binding portion are in Fab format.

8. The polypeptide complex according to any one of claims 1 to 7, wherein the polypeptide complex further comprises an immunoglobulin constant region, for example, an IgG constant region, for example, a human IgG1, IgG4, IgG2, or IgG3 Fc region or a variant thereof.

9. The polypeptide complex according to claim 8, wherein the above-mentioned human IgG Fc region is a human IgG1 Fc region or a variant thereof.

10. The polypeptide complex according to claim 9, wherein the above-mentioned variant comprises one or more substitutions for regulating receptor binding or effector function, promoting dimerization, preventing glycosylation, and / or extending its half-life.

11. The above polypeptide complex is (a) The TIGIT coupling portion is operably connected to the Fc region, and the Fc region is operably connected to the PVRIG coupling portion, (b) The TIGIT coupling portion is operably connected to the PVRIG coupling portion, and the PVRIG coupling portion is operably connected to the Fc region, or (c) The polypeptide complex according to any one of claims 8 to 10, wherein the PVRIG binding portion is operably connected to the TIGIT binding portion, and the TIGIT binding portion is operably connected to the Fc region.

12. A polypeptide complex according to any one of claims 1 to 11, comprising one, two or more TIGIT binding sites and one, two or more PVRIG binding sites.

13. The polypeptide complex according to claim 12, wherein the TIGIT binding portions are the same or different, and / or the PVRIG binding portions are the same or different.

14. It comprises two heavy chains and two light chains, the TIGIT binding portion is in Fab format, the PVRIG binding portion is in scFv format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain domains that are operablely linked to be in the format VH1-CH1-Fc-scFv or scFv-VH1-CH1-Fc, The first light chain and the second light chain each include domains that are operablely linked to be in the VL1-CL format, The polypeptide complex according to any one of claims 1 to 13, wherein the above-mentioned VH1-CH1 and VL1-CL are derived from the TIGIT binding site, and the above-mentioned scFv is derived from the PVRIG binding site.

15. It comprises two heavy chains and two light chains, the TIGIT binding portion is in scFv format, the PVRIG binding portion is in Fab format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each include domains that are operablely linked to be in the format scFv-VH2-CH1-Fc or VH2-CH1-Fc-scFv, The first light chain and the second light chain each include domains that are operablely linked to be in the VL2-CL format, The polypeptide complex according to any one of claims 1 to 13, wherein the scFv is derived from the TIGIT binding site, and the VH2-CH1 and VL2-CL are derived from the PVRIG binding site.

16. It comprises two heavy chains and four light chains, and the TIGIT binding portion and the PVRIG binding portion are in Fab format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each include domains that are operablely linked to be in the format VH1-CH1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-CH1, VH1-CH1-VH2-CH1-Fc, or VH2-CH1-VH1-CH1-Fc. The first light chain and the second light chain each include domains that are operablely linked to be in the VL1-CL format, The third and fourth light chains each include domains that are operablely linked to be in the VL2-CL format, The polypeptide complex according to any one of claims 1 to 13, wherein the above-mentioned VH1-CH1 and VL1-CL are derived from the TIGIT binding site, and the above-mentioned VH2-CH1 and VL2-CL are derived from the PVRIG binding site.

17. It comprises two heavy chains and four light chains, and the TIGIT binding portion and the PVRIG binding portion are in Fab format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain domains that are operablely linked to be in the format VH1-C1-Fc-VH2-CH1, VH2-CH1-Fc-VH1-C1, VH1-C1-VH2-CH1-Fc, or VH2-CH1-VH1-C1-Fc, The first light chain and the second light chain each include domains that are operablely linked to be in the VL1-C2 format, The third and fourth light chains each include domains that are operablely linked to be in the VL2-CL format, The polypeptide complex according to any one of claims 1 to 13, wherein VH1-C1 and VL1-C2 are derived from the TIGIT binding site, and VH2-CH1 and VL2-CL are derived from the PVRIG binding site.

18. It comprises two heavy chains and four light chains, and the TIGIT binding portion and the PVRIG binding portion are in Fab format, and from the N-terminus to the C-terminus, The first heavy chain and the second heavy chain each contain domains that are operablely linked to be in the format VH1-CH1-Fc-VH2-C1, VH2-C1-Fc-VH1-CH1, VH1-CH1-VH2-C1-Fc, or VH2-C1-VH1-CH1-Fc. The first light chain and the second light chain each include domains that are operablely linked to be in the VL1-CL format, The third light chain and the fourth light chain each include domains that are operablely linked to be in the VL2-C2 format, The polypeptide complex according to any one of claims 1 to 13, wherein the above-mentioned VH1-CH1 and VL1-CL are derived from the TIGIT binding site, and the above-mentioned VH2-C1 and VL2-C2 are derived from the PVRIG binding site.

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

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

21. The polypeptide complex according to claim 20, wherein the peptide linker is a hinge region or a GS linker, for example, (GS)n, (GGS)n, (GGGS)n, (GGGGGS)n, (GGSG)n, (GGGSSS)n, where n is an integer from 1 to 9.

22. (i) A first heavy chain and a second heavy chain containing Sequence ID No. 17, and a first light chain and a second light chain containing Sequence ID No. 18, or (ii) The polypeptide complex according to claim 14 or 15, comprising a first heavy chain and a second heavy chain containing SEQ ID NO: 19, and a first light chain and a second light chain containing SEQ ID NO:

20.

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

24. A vector comprising an isolated nucleic acid molecule as described in 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 a polypeptide complex according to any one of claims 1 to 22 and a pharmaceutically acceptable carrier.

27. A method for producing a polypeptide complex according to any one of claims 1 to 22, A step of culturing host cells containing an expression vector encoding the above polypeptide complex under appropriate conditions, The process of collecting the above polypeptide complex from a cell culture and A method that includes this.

28. A method for treating or preventing cancer or immune-related disorders in subjects (e.g., humans and non-human mammals, e.g., mice), comprising administering an effective amount of a polypeptide complex according to any one of claims 1 to 22 or a pharmaceutical composition according to claim 26 to the subjects.

29. The method according to 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, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system tumors, mesothelioma, myeloma, and sarcoma, for example, the cancer is colon cancer.

30. The method according to claim 28, wherein the above-mentioned immune-related disorder is T-cell dysfunction or infection.

31. The method according to claim 29 or 30, further comprising administering an additional therapeutic agent to the subject described above.

32. A polypeptide complex according to any one of claims 1 to 22, for use in the treatment or prevention of cancer, T-cell dysfunction, or infection.

33. Use of a polypeptide complex according to any one of claims 1 to 22 in the manufacture of a pharmaceutical product for treating or preventing cancer, T-cell dysfunction, or infectious disease.

34. The polypeptide complex 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, stomach cancer, pancreatic cancer, lymphoma, leukemia, uterine cancer, cervical cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, head and neck cancer, germ cell cancer, bone cancer, thyroid cancer, skin cancer, central nervous system tumors, mesothelioma, myeloma, and sarcoma, for example, the cancer is colon cancer.

35. A kit comprising a container containing a polypeptide complex according to any one of claims 1 to 22.

Images