PVRIG / TIGIT bispecific antibody pharmaceutical composition and application thereof

Abstract

Provided are a PVRIG / TIGIT bispecific antibody pharmaceutical composition and a use thereof. Specifically, provided is a pharmaceutical composition comprising a PVRIG / TIGIT bispecific antibody and a buffer solution. Further, the pharmaceutical composition further comprises a sugar and a surfactant. Also provided are pharmaceutical compositions for the treatment of cancer or infectious diseases.

Description

A PVRIG / TIGIT bispecific antibody pharmaceutical composition and its use

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on June 8, 2022, with application number 202210641617.6 and invention name “A PVRIG / TIGIT bispecific antibody pharmaceutical composition and its use”, the entire contents of which are incorporated by reference into this disclosure. Technical Field

[0002] The present invention relates to the field of pharmaceutical preparations, and in particular, to a pharmaceutical composition comprising a PVRIG / TIGIT bispecific antibody or an antigen-binding fragment thereof. Background Art

[0003] The basis of immunotherapy is to manipulate and / or regulate the immune system, including both innate and adaptive immune responses, with the general goal of treating diseases by controlling the immune response to "foreign agents" (such as pathogens or tumor cells). The immune system is a highly complex system composed of numerous cell types, and these cells have intricate and subtle systems that control those interactions and responses. The concept of cancer immune surveillance is based on the theory that the immune system can recognize tumor cells, initiate immune responses, and inhibit tumor development and / or progression. However, it is clear that many cancer cells have developed mechanisms to evade the immune system, which can allow unchecked tumor growth. Cancer / tumor immunotherapy focuses on the development of new and novel agonists and / or antagonists that can activate and / or activate the immune system to achieve a more effective anti-tumor response, enhance the killing of tumor cells and / or inhibit tumor growth.

[0004] In recent years, immune checkpoint therapy targeting immune cell co-inhibitory receptors has made great progress in tumor immunotherapy, and the discovery and validation of new co-inhibitory receptors has become a global competitive hotspot. PVRIG is expressed on NK cells and T cells and has several similarities with other known immune checkpoints. When PVRIG binds to its ligand (PVRL2), it triggers an inhibitory signal that weakens the immune response of NK cells and T cells against target cells (i.e., similar to PD-1 / PD-L1). Blocking the binding of PVRL2 to PVRIG cuts off this inhibitory signal of PVRIG and thereby modulates the immune response of NK cells and T cells. Similarly, TIGIT is another target of interest, and binding to its cognate ligand PVR has been shown to directly inhibit the cytotoxicity of NK cells and T cells through its intracellular ITIM domain. Knockout of the TIGIT gene or blocking antibodies to the TIGIT / PVR interaction have been shown to enhance NK cell killing in vitro or exacerbate autoimmune diseases in vivo.

[0005] TIGIT and PVRIG belong to the DNAM superfamily and have been shown to be co-expressed in a variety of tumor-infiltrating lymphocytes to exert immunosuppressive effects. Bispecific antibodies that can simultaneously target PVRIG and TIGIT have potential synergistic effects and are an attractive therapeutic approach for single antibody therapy.

[0006] Antibodies are relatively high-molecular-weight proteins. Because proteins are relatively unstable in solution and are prone to particle formation and aggregation, stability is a major challenge in the development of large-molecule protein drugs. Consequently, there is a need for stable formulations of PVRIG / TIGIT bispecific antibodies for pharmaceutical applications, such as the treatment of various cancers and infectious diseases. Such formulations exhibit excellent stability at room temperature or elevated temperatures, potentially reducing the transportation and storage costs of antibody drugs.

[0007] Summary of the Invention

[0008] The present invention discloses a PVRIG / TIGIT bispecific antibody pharmaceutical composition and uses thereof.

[0009] In one aspect, the present invention provides a pharmaceutical composition comprising:

[0010] (i) a bispecific antibody or an antigen-binding fragment thereof, comprising binding domains that specifically bind to PVRIG and TIGIT,

[0011] (ii) buffer,

[0012] (iii) non-reducing sugars, and

[0013] (iv) nonionic surfactants,

[0014] Wherein, the bispecific antibody or antigen-binding fragment thereof comprises:

[0015] (a) a first antigen-binding portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL form an anti-TIGIT antigen-binding domain; wherein the TIGIT VH comprises the HCDR1, HCDR2, and HCDR3 of the VH of any one of SEQ ID NOs: 12 or 14; and wherein the TIGIT VL comprises the LCDR1, LCDR2, and LCDR3 of the VL of any one of SEQ ID NOs: 11 or 13;

[0016] (b) a second antigen-binding portion comprising a VHH that specifically binds to PVRIG, said VHH comprising CDR1, CDR2 and CDR3 of any one of SEQ ID NOs: 9 or 10.

[0017] Preferably, the first antigen binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequence:

[0018] (1) SEQ ID NO: 21, 22, 23, 27, 28 and 29, respectively; or

[0019] (2) SEQ ID NOs: 24, 25, 26, 30, 31 and 32, respectively; or

[0020] (3) a sequence having at least 90% identity with the sequence shown in (1) to (2) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions;

[0021] The second antigen binding portion comprises CDR1, CDR2 and CDR3 of the following sequence:

[0022] (1) SEQ ID NOs: 15, 16 and 17, respectively; or

[0023] (2) SEQ ID NOs: 18, 19, and 20, respectively; or

[0024] (3) A sequence having at least 90% identity with the sequence shown in (1) to (2) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

[0025] Preferably, the VH of the first antigen-binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 12 or 14; the VL of the first antigen-binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 11 or 13; and the second antigen-binding portion comprises a sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 9 or 10.

[0026] In some embodiments, in any of the foregoing pharmaceutical compositions, the concentration of the PVRIG / TIGIT bispecific antibody or its antigen-binding fragment is about 10 mg / ml to about 200 mg / ml, preferably, about 10 mg / ml to about 150 mg / ml, about 15 mg / ml to about 120 mg / ml, about 20 mg / ml to about 100 mg / ml, about 20 mg / ml to about 80 mg / ml, about 20 mg / ml to about 70 mg / ml, or about 30 mg / ml to about 60 mg / ml.

[0027] In some embodiments, in any of the foregoing pharmaceutical compositions, the buffer is selected from acetic acid-sodium acetate buffer or histidine-histidine hydrochloride buffer, preferably, the buffer concentration is about 10 mM to about 80 mM, about 15 mM to about 70 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, or about 20 mM to about 30 mM.

[0028] In some embodiments, in any of the foregoing pharmaceutical compositions, the non-reducing sugar is selected from sucrose, sorbitol or trehalose, and preferably, the concentration of the non-reducing sugar is about 6% to about 10% (w / v), about 6% to about 9% (w / v), about 7% to about 9% (w / v), or about 7% to about 8% (w / v).

[0029] In some embodiments, in any of the foregoing pharmaceutical compositions, the nonionic surfactant is polysorbate 80, preferably, the concentration of the nonionic surfactant is about 0.01% to about 0.10% (w / v), about 0.01% to about 0.08% (w / v), about 0.02% to about 0.07% (w / v), or about 0.02% to about 0.06% (w / v).

[0030] In some embodiments, any of the aforementioned pharmaceutical compositions is an injection, preferably a subcutaneous injection, an intravenous injection, or an intravenous infusion.

[0031] In some embodiments, any of the foregoing pharmaceutical compositions comprises:

[0032] (i) about 10 mg / ml to about 200 mg / ml of a PVRIG / TIGIT bispecific antibody or antigen-binding fragment thereof;

[0033] (ii) about 10 mM to about 100 mM acetic acid-sodium acetate buffer;

[0034] (iii) about 6% to about 10% (w / v) sucrose;

[0035] (iv) about 0.01% to about 0.10% (w / v) polysorbate 80.

[0036] Preferably, the pharmaceutical composition comprises about 50 mg / mL of PVRIG / TIGIT bispecific antibody or antigen-binding fragment thereof, 20 mM acetic acid-sodium acetate buffer, about 8% w / v sucrose, and about 0.02% polysorbate 80.

[0037] Preferably, the pharmaceutical composition comprises about 50 mg / mL of PVRIG / TIGIT bispecific antibody or antigen-binding fragment thereof, 20 mM acetic acid-sodium acetate buffer, about 8% w / v sucrose, and about 0.04% polysorbate 80.

[0038] Preferably, the pharmaceutical composition comprises about 50 mg / mL of PVRIG / TIGIT bispecific antibody or antigen-binding fragment thereof, 20 mM acetic acid-sodium acetate buffer, about 8% w / v sucrose, and about 0.06% polysorbate 80.

[0039] In some embodiments, the pH of any of the foregoing pharmaceutical compositions is about 4.5-5.5, preferably, about 5.0-5.2.

[0040] In some embodiments, any of the foregoing pharmaceutical compositions are stable at 2°C to 8°C for at least 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, or 6 months.

[0041] In some embodiments, any of the foregoing pharmaceutical compositions are stable at 25°C for at least 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, or 6 months.

[0042] In some embodiments, any of the foregoing pharmaceutical compositions are stable at 40°C for at least 1 week, 2 weeks, 3 weeks, or 4 weeks.

[0043] In another aspect, any one of the above pharmaceutical compositions is used for preparing a medicament for treating cancer or infectious diseases; wherein the cancer is selected from solid tumors and blood tumors.

[0044] In some embodiments, the pharmaceutical composition is used in combination with another therapeutic agent or with surgery; wherein the additional therapeutic agent is selected from radiation therapy, chemotherapy, an oncolytic drug, a cytotoxic agent, a cytokine, an immunostimulatory antibody, an immunomodulatory drug, an activator of a co-stimulatory molecule, an inhibitor of an inhibitory molecule, a vaccine or a cellular immunotherapy, and the surgery is surgery.

[0045] In another aspect, the present invention provides a method for treating cancer or infectious diseases, comprising administering an effective amount of any of the aforementioned pharmaceutical compositions to a patient in need thereof, wherein the cancer is selected from solid tumors and hematological tumors.

[0046] In another aspect, the present invention provides any of the aforementioned pharmaceutical compositions for use in treating cancer or infectious diseases; wherein the cancer is selected from solid tumors and blood tumors.

[0047] Definitions and Explanations of Terms

[0048] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art. For terms that are clearly defined herein, the meaning of the term shall be based on the definitions.

[0049] Furthermore, unless otherwise indicated herein, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly indicated otherwise.

[0050] The terms "comprising," "including," and "having" are used interchangeably herein to indicate the inclusiveness of a solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "comprising," "including," and "having" in this document also provides for "consisting of" solutions. For example, "a composition comprising A and B" should be understood to include the following technical solutions: a composition consisting of A and B, as well as a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition."

[0051] The term "and / or" as used herein includes the meanings of "and," "or," and "all or any other combination of elements linked by the associated term."

[0052] When "about" is used before a numerical value, it indicates that the value may fluctuate above or below the value, with an optional fluctuation range of 10%. For example, "about 10%" means "9% to 11%." When "about" is used before a numerical range, it indicates that the value may fluctuate above or below the endpoints of the range, with the upper endpoint fluctuating upward and the lower endpoint fluctuating downward. For example, "about 10 to 20 nM" means the range is "9 to 22 nM." Optionally, the "about" fluctuation range can also fluctuate above or below 5%, 4%, 2%, or 1%.

[0053] The terms "T cell immunoreceptor having Ig and ITIM domains," "TIGIT," "TIGIT antigen," "Vstm3," and "WUCAM" are used interchangeably and include various mammalian isoforms, such as human Tigit, orthologs of human Tigit, and analogs comprising at least one epitope within Tigit, as well as analogs having at least one epitope shared with TIGIT. The amino acid sequence of TIGIT (e.g., human TIGIT), and the nucleotide sequence encoding the same, are known in the art.

[0054] The term "PVRIG" or "PVRIG protein" herein may optionally include any such protein or variant, conjugate, or fragment thereof, including but not limited to known or wild-type PVRIG as described herein, as well as any naturally occurring splice variants, amino acid variants, or isoforms, and in particular, the ECD fragment of PVRIG. "Anti-PVRIG antibodies" (including antigen-binding fragments) that bind to PVRIG and prevent activation by PVRL2 (e.g., most often by blocking the interaction between PVRIG and PVLR2) are useful for enhancing T cell and / or NK cell activation and for treating diseases such as cancer and pathogen infection.

[0055] The PVRIG / TIGIT bispecific antibody of the present invention specifically binds to human TIGIT, and preferably the ECD of human TIGIT, and PVRIG, and more preferably the ECD of human PVRIG.

[0056] The term "specific binding" herein refers to the ability of an antigen-binding molecule (e.g., an antibody) to specifically bind to an antigen and substantially the same antigen, typically with high affinity, but not to bind to unrelated antigens with high affinity. Affinity is typically measured as an equilibrium dissociation constant (KD), where a lower KD indicates a higher affinity. For example, a high affinity antibody typically refers to an affinity of approximately 1 × 10 -7 M or lower, about 1×10 -8 M or lower, about 1×10 -9 M or less, about 1×10 -10 M or less, 1×10 -11 M or lower or 1×10 -12 The KD is calculated as follows: KD = Kd / Ka, where Kd represents the off-rate and Ka represents the on-rate. The equilibrium dissociation constant, KD, can be measured using methods known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.

[0057] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0058] The term "antibody" is used in the broadest sense herein to refer to a polypeptide or combination of polypeptides that comprises sufficient sequence from an immunoglobulin heavy chain variable region and / or sufficient sequence from an immunoglobulin light chain variable region to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which comprise mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0059] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0060] "Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, fragments of whole antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0061] The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or produced during the production of the preparation, such variants typically being present in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.

[0062] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, diabodies, and single-domain antibodies.

[0063] The term "bispecific" herein refers to a molecule having at least two antigen-binding sites, i.e., at least "bispecific," and may be "trispecific," "tetraspecific," etc.; each of the at least two antigen-binding sites binds to a different epitope of the same antigen or to different epitopes of different antigens. Alternatively, a "bispecific" may be an antibody-derived molecule having at least two antigen-binding sites, such as an immunoconjugate.

[0064] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0065] The term "variable region" herein refers to the region of an antibody heavy or light chain that is involved in binding the antibody to an antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, each comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0066] For further description of CDRs, see Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, using tool websites including but not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0067] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0068] The term "conservative amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). For example, the amino acids within each of the following groups are conservative amino acid residues of each other, and substitutions of amino acid residues within the group are substitutions of conservative amino acids:

[0069] Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0070] 1) Alanine (A), serine (S), threonine (T);

[0071] 2) Aspartic acid (D), glutamic acid (E);

[0072] 3) Asparagine (N), glutamine (Q);

[0073] 4) Arginine (R), Lysine (K), Histidine (H);

[0074] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0075] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0076] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0077] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0078] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0079] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0080] Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention (SEQ ID NO: 1). BLAST protein searches can be performed with the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. In order to obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0081] The term "pharmaceutical composition" herein refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that have unacceptable toxicity to the subject to which the pharmaceutical composition is administered. The purpose of the pharmaceutical composition is to maintain the stability of the antibody active ingredient, promote administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity. Herein, "pharmaceutical composition" and "preparation" are not mutually exclusive. In some embodiments, the term "pharmaceutical composition" herein includes antibodies, antibody-derived molecules such as immunoconjugates.

[0082] The term "immunoconjugate" herein refers to a polypeptide molecule comprising at least one effector molecule and at least one antibody or a functional fragment thereof.

[0083] The term "effector molecule" herein is a portion of an immunoconjugate that is intended to have a desired effect on the cells to which the immunoconjugate is targeted. Effector molecules are also referred to as effector moieties (EMs), therapeutic agents, diagnostic agents, tracers, or similar terms.

[0084] The term "stable" herein refers to a formulation in which the protein substantially maintains its physical stability and / or chemical stability and / or biological activity upon storage. The shelf life is generally selected based on the intended shelf life of the pharmaceutical composition. Various analytical techniques are available in the art for measuring protein stability.

[0085] A stable pharmaceutical antibody formulation is one in which no significant changes are observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, and more preferably 1 year. Additionally, stable liquid formulations include those that exhibit desired characteristics after storage at temperatures including 25°C and 40°C for periods of 1 month and 3 months. Stable formulations are, for example, colorless or clear to slightly opalescent by visual analysis. The concentration, pH, and osmolality of the formulation vary by no more than ±10%. Typically, no more than about 10%, preferably no more than about 5%, of truncation is observed. Typically, no more than about 10%, preferably no more than about 5%, of aggregation is formed.

[0086] An antibody "retains its physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS).

[0087] An antibody "retains its chemical stability" in a pharmaceutical formulation if it shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0088] An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody can be determined, for example, by antigen binding assays.

[0089] The term "buffer" herein encompasses agents that maintain the pH of solutions of the pharmaceutical compositions of the present invention within an acceptable range, including acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0090] The term "non-reducing sugar" herein may include at least one selected from trehalose or sucrose.

[0091] The term "nonionic surfactant" herein is selected from the group consisting of nonionic water-soluble monoglycerides, nonionic water-soluble diglycerides, nonionic water-soluble triglycerides, nonionic water-soluble polyethylene glycol monofatty acid esters, nonionic water-soluble polyethylene glycol difatty acid esters, nonionic water-soluble sorbitol fatty acid esters, nonionic polyglycosylated glycerides, nonionic water-soluble triblock copolymers, and combinations thereof. In some embodiments, the nonionic surfactant is polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

[0092] The term "Tm value" in this article refers to the thermal denaturation temperature of the protein, that is, the temperature at which half of the protein unfolds. At this time, the spatial structure of the protein is destroyed. Therefore, the higher the Tm value, the higher the thermal stability of the protein.

[0093] The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject of treatment, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of the extent of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Subjects in need of treatment include subjects already suffering from a condition or disease, as well as subjects susceptible to a condition or disease, or subjects intending to prevent a condition or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, and alleviating, their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0094] The term "patient" herein refers to an organism receiving treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (e.g., monkeys), or non-primate mammals receiving treatment for a disease or condition.

[0095] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0096] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the terms "tumor" or "neoplasm" refer to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein. DETAILED DESCRIPTION

[0097] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0098] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0099] Glossary:

[0100] Unless otherwise specified, the ELISA Binding assay described below uses the following method:

[0101] The PVRIG / TIGIT bispecific antibody specifically binds to hTIGIT-mFc at one end and hPVRIG-His at the other. A 96-well plate was pre-coated with antigen (hTIGIT-mFc) (Manufacturer: Acro, Catalog No.: TIT-H5253). The PVRIG & TIGIT bispecific antibody, diluted to varying concentrations, was added to bind to the antigen on the plate. Unbound components were removed by washing, and then the primary antibody (hPVRIG-His) (Manufacturer: Acro, Catalog No.: PVG-H52H4) was added. After incubation, the plate was washed to remove unbound primary antibody, and then the secondary antibody (His Tag Horseradish Peroxidase-conjugated Antibody) (Manufacturer: R&D, Catalog No.: MAB050H) was added for incubation. After incubation, the plate was washed to remove unbound secondary antibody. Finally, TMB was added for color development, and the reaction was terminated with stop solution. OD values ​​were read using a microplate reader.

[0102] Example 1 Construction, expression and purification of PVRIG / TIGIT bispecific antibody

[0103] 1.1 Construction of PVRIG / TIGIT bispecific antibody

[0104] Two humanized anti-PVRIG VHH antibodies (PVRIG-A50-H1b, PVRIG-A105-H1) and two humanized anti-TIGIT monoclonal antibodies (TIGIT-002-H4L3, TIGIT-005-H2L1d) were generated. The anti-PVRIG VHH antibodies were linked to the N-terminus of the heavy chains of the anti-TIGIT humanized antibodies using a G4S linker peptide to generate anti-PVRIGxTIGIT humanized bispecific antibodies, designated LC-BsAb-002, LC-BsAb-006, LC-BsAb-009, and LC-BsAb-010, respectively. Table 1 shows the sequences of the heavy chain fusion polypeptides (HC) and light chain polypeptides (LC) of the four bispecific antibodies, Table 2 shows the variable region sequences of the bispecific antibodies, and Table 3 shows the CDR sequences of the bispecific antibodies according to the Kabat classification.

[0105] Table 1 Bispecific antibody fusion polypeptide sequences

[0106] Table 2 Variable region sequences of bispecific antibodies

[0107] Table 3 KABAT analysis results of bispecific antibodies

[0108] 1.2 Expression of PVRIG / TIGIT bispecific antibody

[0109] The PVRIG / TIGIT bispecific antibody gene was transfected into CHO-K1 cells using genetic engineering techniques. High-yielding clones were selected through MSX pressure screening and subcultured in CD CHO medium. The PVRIG / TIGIT bispecific antibody was expressed using a fed-batch culture system. The basal medium used was Optima's CHO CDP9, and the culture cycle lasted 14-16 days. The reactor parameters were pH 6.6-7.2, dissolved oxygen (DO) ≥20%, a rotation speed of 75-80 RPM, and an initial culture temperature of 36.0-37.0°C. On day 6 of culture, the temperature was lowered to 33.5-34.5°C until harvest.

[0110] Feed media were Optima's XF04 and CD FS08, which were fed every other day starting from Day 3, with feed volumes of 5% and 0.5% of the volume, respectively, until harvest.

[0111] 1.3 Purification of PVRIG / TIGIT bispecific antibody

[0112] The PVRIG / TIGIT bispecific antibody is purified using a multi-step chromatography, concentration, and filtration unit operation. The supernatant harvest is captured by Protein A affinity chromatography (AT ProteinA Diamond Plus). The captured antibody solution is incubated at low pH to inactivate potential viruses. After neutralization, the antibody solution is deep filtered to remove the precipitate. Anion exchange chromatography (Diamond Q) is then performed to remove impurities such as HCD, HCP, and shed Protein A, and cation exchange (Diamond S) chromatography is performed to remove impurities such as HCP and aggregates. Filtration is performed through a nanofiltration membrane pack to remove potential endogenous and exogenous viruses. The antibody solution is then concentrated using an ultrafiltration membrane pack and the buffer is exchanged to complete the purification process, obtaining the PVRIG / TIGIT bispecific antibody protein stock solution.

[0113] 1.4 BIAcore Detection of the Affinity of PVRIG / TIGIT Bispecific Antibody to Human, Cynomolgus Monkey, and Mouse TIGIT and PVRIG Proteins

[0114] This experiment used a Protein A chip, and the time required for the chip to capture the diluted antibody was measured manually (manual run) to achieve saturated antigen binding Rmax of 50RU. Human, cynomolgus monkey, and mouse TIGIT and PVRIG proteins were gradiently diluted to 20, 10, 5, 2.5, and 1.25nM. The affinity of the antibody to the antigen was measured using multi-cycle kinetics. In each cycle, after the antibody was injected, gradient concentrations of human, cynomolgus monkey, and mouse TIGIT and PVRIG proteins were injected to allow the antigen to bind and dissociate. After each cycle, the Protein A chip was regenerated with Glycine pH 1.5 (to remove the protein on the chip). BIAcore T200 analysis software was used to fit the affinity KD of the antibody and antigen. The results in Table 4 show that the two bispecific antibodies bind specifically to human and cynomolgus monkey TIGIT and PVRIG proteins with high affinity levels, but do not bind to mouse TIGIT and PVRIG proteins.

[0115] Table 4 Affinity of PVRIG / TIGIT bispecific antibodies to TIGIT and PVRIG proteins from different species

[0116] 1.5 BIAcore detection of co-binding of PVRIG / TIGIT bispecific antibody to human TIGIT and PVRIG protein

[0117] BIAcore was used to characterize the simultaneous binding of bispecific antibodies to two antigens. Antibodies LC-BsAb-002 and LC-BsAb-006 were first captured using a Protein A chip. TIGIT and PVRIG his-tagged proteins were then injected, followed by sequential injections of TIGIT and PVRIG, and PVRIG and TIGIT. Binding signals were recorded, and the chip was regenerated using Glycine pH 1.5. The mobile phase consisted of HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) at a flow rate of 30 μL / min. The binding time for each antigen was 300 s, with a regeneration time of 30 s. The assay temperature was 25°C, and the assay concentrations for hTIGIT and hPVRIG were 20 nM and 50 nM, respectively. The data were analyzed using BIAcore 8K analysis software (version 2.0). The antibody capture level and binding responses (RU) for different antigens were recorded. The stoichiometric ratio of antigen-antibody molecules was calculated based on the molecular weight of the antigen and antibody, providing a preliminary estimate of how many antigens a single antibody molecule can bind. To confirm the interaction between the antibody LC-BsAb-002 and the antigens TIGIT and PVRIG, the following four-step assay was performed: binding to hTIGIT alone, binding to hPVRIG alone, binding to hTIGIT first and then to hPVRIG, and binding to hPVRIG first and then to hTIGIT, with saturation of each antigen.The antibody-antigen binding curves were collected, and the capture levels of the two bispecific antibodies and the binding signals of TIGIT and PVIRIG in each experiment were recorded. The stoichiometric ratio of antigen-antibody molecules was calculated based on this. As shown in Table 5, the binding signals generated by continuous injection of TIGIT and PVIRG were almost the same as those generated by injection of TIGIT and PVIRG alone; and the binding signals generated by continuous TIGIT and PVIRG in the forward and reverse directions were also almost the same, indicating that LC-BsAb-002 and LC-BsAb-006 can bind to hTIGIT and hPVRIG at the same time, and there is no mutual influence between the two antigens; considering the molecular weight of the antibody and antigen, the binding signals generated by continuous injection of TIGIT and PVIRG are almost the same. Based on the amount of TIGIT, the antibody capture level, and the antigen binding level, we preliminarily estimated the stoichiometric ratios of TIGIT to LC-BsAb-002 to be 1.76; the stoichiometric ratios of TIGIT to LC-BsAb-006 to be 1.86; the stoichiometric ratios of PVIRIG to LC-BsAb-002 to be 2.14, and the stoichiometric ratios of PVIRIG to LC-BsAb-006 to be 2.18. The stoichiometric ratios of the two antigen-antibodies are all close to 2. Taking into account the errors caused by the detection method, we speculate that one LC-BsAb-002 or one LC-BsAb-006 bispecific antibody molecule can bind to two TIGIT molecules and two PVRIG molecules at the same time.

[0118] Table 5 BIAcore binding of PVRIG / TIGIT bispecific antibody to TIGIT and PVRIG protein

[0119] 1.6 Killing effect of PVRIG / TIGIT bispecific antibody

[0120] PVRIG / TIGIT bispecific antibodies LC-BsAb-002 and LC-BsAb-006 can effectively promote NK cell killing of target cell WIDR, and can also show concentration-dependent ADCC killing of Treg cells.

[0121] A375 cells were inoculated into mice, and when the tumor grew to an appropriate size, the PVRIG / TIGIT bispecific antibodies LC-BsAb-002 and LC-BsAb-006 were administered. The results showed that the bispecific antibodies had a significant inhibitory effect on A375 tumor growth.

[0122] Example 2 Screening of pH / buffer system

[0123] 2.1 Research Plan

[0124] (1) Design 12 different buffer formulations F1-F12. The specific information of the candidate formulations is shown in Table 1. (2) The PVRIG / TIGIT bispecific antibody LC-BsAb-002 protein stock solution obtained in Example 1 was replaced into F1-F12 using Millipore ultrafiltration and concentration centrifuge tubes, and the protein concentration was adjusted to 50 mg / mL. (3) Accelerated stability tests at 25°C and 40°C were performed to comprehensively investigate the stability of the PVRIG / TIGIT bispecific antibody in the 12 candidate formulations, and the optimal pH / buffer system was screened for the next step of excipient development. The evaluation indicators included appearance, pH, protein concentration, dynamic light scattering (DLS), thermal stability (DSF), full-column imaging capillary isotropic focusing electrophoresis (iCIEF), purity (size exclusion chromatography (SE-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS (NR&R)) and binding activity (ELISA-Binding). The evaluation plan is detailed in Table 6.

[0125] Table 6 pH / buffer system screening prescription and screening investigation plan Note: X = appearance, pH, protein concentration, DLS, iCIEF, SE-HPLC, CE-SDS (NR&R); Y = ELISA-Binding; Z = DSF. T0 = starting point of the study, W = week, same below.

[0126] 2.2 Screening results

[0127] The main results of pH / buffer system screening are summarized in Tables 7 to 16 below:

[0128] Table 7 pH / buffer system screening appearance test results

[0129] Table 8 pH / buffer system screening pH test results

[0130] Table 9 pH / buffer system screening protein concentration (mg / mL) test results

[0131] Table 10 pH / buffer system screening DLS test results (diameter, nm)

[0132] Table 11 pH / buffer system screening T0 sampling point DSF (℃) test results

[0133] Table 12 pH / buffer system screening iCIEF (%) detection results Note: MP: Main Peak; AP: Acidic Peak; BP: Basic Peak

[0134] Table 13 pH / buffer system screening SE-HPLC (%) detection results Note: MP: Main Peak, HMW: High Molecular Weight, high polymer; LMW: Low Molecular Weight, low polymer; ND: Not Detected, not detected

[0135] Table 14 pH / buffer system screening CE-SDS-NR (%) detection results Note: MP: Main Peak; HMW: High Molecular Weight; LMW: Low Molecular Weight; ND: Not Detected

[0136] Table 15 pH / buffer system screening CE-SDS-R (%) detection results Note: LC+HC: light chain + heavy chain; Other: other

[0137] Table 16 pH / buffer system screening ELISA-Binding (%) test results

[0138] 2.3 Results Analysis

[0139] (1) Appearance: The appearance of the acetic acid system is better than that of the histidine, citric acid and phosphate buffer systems.

[0140] (2) pH value: The pH value of all buffer systems did not change significantly during the entire investigation period.

[0141] (3) Protein concentration: The protein concentration of all buffer systems did not change significantly during the entire investigation period.

[0142] (4) Thermal stability DSF: There was no significant difference in the Tm values ​​of all buffer systems at T0.

[0143] (5) DLS: The particle size of the acetate buffer system is smaller than that of other buffer systems, among which F1 <F2<F3。

[0144] (6) iCIEF: The main peaks of charge isomers in the histidine buffer system and the acetate buffer system were significantly better than those in the citric acid and phosphoric acid systems.

[0145] (7) SE-HPLC: There was no significant difference in monomer purity between the acetic acid and histidine buffer systems. When the samples were stored at 40°C for 4 weeks, the monomer purity of the citric acid and phosphoric acid systems was significantly lower than that of the acetic acid and histidine buffer systems.

[0146] (8) CE-SDS (NR): When the sample was stored at 40°C for 4 weeks, the F2 formulation had the highest monomer main peak ratio and the highest protein purity.

[0147] (9) CE-SDS(R): Comparing the ratio of the main monomer peak, the protein purity of the acetate and histidine buffer system was better than that of the citric acid and phosphoric acid system, while there was no significant difference in the acetate and histidine buffer system.

[0148] (10) ELISA-Binding: There was no significant difference in binding activity among all buffer systems (Note: The normal fluctuation range of ELISA is 70-130).

[0149] Summary: A comprehensive evaluation of the various test parameters of 12 candidate formulations revealed that the acetic acid and histidine buffer systems performed significantly better than the citric acid and phosphoric acid systems. The acetate buffer system also slightly outperformed the histidine buffer system in DLS and CE-SDS(R) performance. Formulation F2 (20 mM acetic acid-sodium acetate, pH 5.0) performed well in appearance, particle size, and purity, and exhibited slightly superior stability compared to the other formulations. Therefore, formulation F2 was selected for subsequent excipient screening.

[0150] Example 3 Screening of auxiliary materials

[0151] 3.1 Research Plan

[0152] (1) Based on the optimal pH / buffer system (20 mM acetic acid-sodium acetate pH 5.0) screened in Example 2, 6 different excipients were added. The specific information of the candidate formulations is shown in Table 12.

[0153] (2) The stability of the PVRIG / TIGIT bispecific antibody LC-BsAb-002 (protein concentration 50 mg / mL) in six candidate formulations was comprehensively investigated through stability tests, shaking tests, and freeze-thaw tests to screen the optimal excipient for the next step of surfactant development. The evaluation criteria included: appearance, pH, protein concentration, dynamic light scattering (DLS), thermal stability (DSF), osmotic pressure, iCIEF, SE-HPLC, CE-SDS (NR&R), and binding activity (ELISA-Binding). The evaluation plan is detailed in Table 17.

[0154] Table 17 Excipient screening prescription and screening investigation plan Note: X = appearance, pH, protein concentration, DLS, iCIEF, SE-HPLC, CE-SDS (NR&R); Y = ELISA-Binding, microparticulate matter index (MFI); Z = DSF, osmotic pressure. *All % in the table refer to % (w / v). a F / T: Freeze / Thaw. M = month, D = day, C = cycle, same below.

[0155] 3.2 Screening results

[0156] The main results of excipient screening are summarized in Tables 18 to 32 below:

[0157] Table 18 Appearance test results of auxiliary materials screening

[0158] Table 19 pH test results of excipient screening

[0159] Table 20 Excipient screening protein concentration (mg / mL) test results

[0160] Table 21 Excipient screening DLS (diameter, nm) test results

[0161] Table 22 Excipient screening insoluble particles (MFI, particles / mL) test results ≥10: particles with a diameter ≥10 μm; ≥25: particles with a diameter ≥25 μm.

[0162] Table 23 Excipient screening T0 sampling point DSF (℃) and osmotic pressure (mOsm / kg) test results

[0163] Table 24 Excipient screening 2-8°C and 25°C stability test iCIEF (%) test results

[0164] Table 25 Excipient screening 40 ° C stability test, shaking test and freeze-thaw test iCIEF (%) test results

[0165] Table 25 continued Note: MP: Main Peak; AP: Acidic Peak; BP: Basic Peak

[0166] Table 26 Excipient screening 2~8℃ and 25℃ stability test SE-HPLC (%) test results

[0167] Table 27 Excipient screening 40°C stability test, shaking test and freeze-thaw test SE-HPLC (%) test results

[0168] Table 27 continued Note: MP: Main Peak; HMW: High Molecular Weight; LMW: Low Molecular Weight; ND: Not Detected

[0169] Table 28 Excipient screening 2-8°C and 25°C stability test CE-SDS-NR (%) test results

[0170] Table 29 Excipient screening 40°C stability test, shaking test and freeze-thaw test CE-SDS-NR (%) test results

[0171] Table 29 continued Note: MP: Main Peak; HMW: High Molecular Weight; LMW: Low Molecular Weight; ND: Not Detected

[0172] Table 30 Excipient screening 2-8°C and 25°C stability test CE-SDS-R (%) test results

[0173] Table 31 Excipient screening 40°C stability test, shaking test and freeze-thaw test CE-SDS-R (%) test results

[0174] Table 31 continued Note: LC+HC: light chain + heavy chain; Other: other

[0175] Table 32 Excipient Screening ELISA-Binding (%) Test Results

[0176] 3.3 Results Analysis

[0177] (1) Appearance: The appearance of the formulas prepared with arginine (F2-2) and sodium chloride (F2-3) as excipients was significantly worse than that of the formulas prepared with other excipients, while there was no significant difference in the appearance of glycine (F2-1), sucrose (F2-4), trehalose (F2-5), and sorbitol (F2-6).

[0178] (2) pH value: There was no significant difference in the pH values ​​of all candidate formulations during the entire investigation period.

[0179] (3) Protein concentration: The protein concentration of all candidate formulations did not change significantly during the entire investigation period.

[0180] (4) Thermal stability DSF: There was no significant difference in the Tm values ​​of all candidate formulations at T0.

[0181] (5) DLS: The particle sizes of the formulations containing arginine (F2-2) and sodium chloride (F2-3) as excipients were larger than those of the formulations containing other excipients, while the particle sizes of glycine (F2-1), sucrose (F2-4), trehalose (F2-5), and sorbitol (F2-6) showed no significant difference.

[0182] (6) Insoluble particles: The sucrose (F2-4) formulation performed slightly better than other excipient formulations in controlling the generation of insoluble particles.

[0183] (7) iCIEF: Taking all the conditions into consideration, the main peak of charge isomers in the formulation with glycine (F2-1) as the excipient was the lowest (for example, when stored at 40°C for 4 weeks and at 25°C for 4 weeks), while there was no significant difference in the formulations with other excipients.

[0184] (8) SE-HPLC: There was no significant difference in the monomer purity of the formulations prepared with sucrose (F2-4), trehalose (F2-5), and sorbitol (F2-6). When stored at high temperature for a long time, the monomer purity of formulations F2-4 to F2-6 was significantly higher than that of formulations F2-1 to F2-3.

[0185] (9) CE-SDS (NR): There was no significant difference in the protein purity of all candidate formulations.

[0186] (10) CE-SDS(R): When the samples were stored at room temperature or high temperature for a long time, the formulation with sucrose (F2-4) as an auxiliary material performed slightly better, while the shaking test and freeze-thaw test F2-1-F2-3 performed slightly better.

[0187] (11) ELISA-Binding: There was no significant difference in the binding activity of all candidate formulations.

[0188] Summary: Through a comprehensive evaluation of all test parameters of the six candidate formulations, formulation F2-4 (20 mM acetic acid-sodium acetate, pH 5.0; 0.04% (w / v) PS80; 8% (w / v) sucrose) performed well compared to the other formulations in terms of appearance, particle size, and purity, and was slightly superior in terms of stability during long-term storage at room temperature or high temperature. Therefore, formulation F2-4 was selected for subsequent surfactant strength screening.

[0189] Example 4 Surfactant Strength Screening

[0190] 4.1 Research Plan

[0191] (1) Based on the optimal pH / buffer system and excipients (20 mM acetic acid-sodium acetate pH 5.0; 8% (w / v) sucrose) screened in Examples 2 and 3, three surfactants (polysorbate 80, PS80) at different concentrations were added. The specific information of the candidate formulations is shown in Table 28. (2) The stability of the PVRIG / TIGIT bispecific antibody LC-BsAb-002 (protein concentration 50 mg / mL) in the three candidate formulations was comprehensively investigated through stability experiments, shaking experiments, and freeze-thaw experiments to screen for the optimal surfactant strength. The evaluation indicators included: appearance, pH, protein concentration, dynamic light scattering (DLS), cation exchange chromatography (CEX), SE-HPLC, CE-SDS (NR), and binding activity (ELISA-Binding). The evaluation plan is detailed in Table 33.

[0192] Table 33 Surfactant strength screening prescription and screening investigation plan Note: X = appearance, pH, protein concentration, DLS, iCIEF, SE-HPLC, CE-SDS (NR); Y = ELISA-Binding, insoluble particulate matter (MFI).

[0193] 4.2 Screening results

[0194] The main results of the surfactant strength screening are summarized in Tables 34 to 45 below:

[0195] Table 34 Surfactant strength screening appearance test results

[0196] Table 35 Surfactant strength screening pH test results

[0197] Table 36 Surfactant Strength Screening Protein Concentration (mg / mL) Test Results

[0198] Table 37 Surfactant Strength Screening DLS (Particle Size, nm) Test Results

[0199] Table 38 Surfactant Strength Screening Insoluble Particles (MFI, Particles / mL) Test Results

[0200] Table 38 continued

[0201] Table 39 Surfactant Strength Screening 2-8°C and 25°C Stability Test CEX (%) Test Results

[0202] Table 39 continued

[0203] Table 40 Surfactant strength screening 40 ° C stability test, shaking test and freeze-thaw test CEX (%) test results

[0204] Table 40 continued Note: MP: Main Peak; AP: Acidic Peak; BP: Basic Peak

[0205] Table 41 Surfactant Strength Screening 2-8°C and 25°C Stability Test SE-HPLC (%) Test Results

[0206] Table 41 continued

[0207] Table 42 Surfactant strength screening 40 ° C stability test, shaking test and freeze-thaw test SE-HPLC (%) test results

[0208] Table 42 continued Note: MP: Main Peak; HMW: High Molecular Weight; LMW: Low Molecular Weight; ND: Not Detected

[0209] Table 43 Surfactant strength screening 2~8℃ and 25℃ stability test CE-SDS-NR (%) test results

[0210] Table 43 continued

[0211] Table 44 Surfactant strength screening 40 ° C stability test, shaking test and freeze-thaw test CE-SDS-NR (%) test results

[0212] Table 44 continued Note: MP: Main Peak, main peak; HMW: High Molecular Weight, high polymer; LMW: Low Molecular Weight, low polymer

[0213] Table 45 Surfactant Strength Screening ELISA-Binding (%) Test Results

[0214] 4.3 Results Analysis

[0215] (1) Appearance: The appearance of all candidate formulations was consistent and showed no significant changes during the entire investigation period.

[0216] (2) pH value: The pH values ​​of all candidate formulations did not change significantly during the entire investigation period.

[0217] (3) Protein concentration: The protein concentration of all candidate formulations did not change significantly during the entire investigation period.

[0218] (4) Insoluble particles: According to the quality standards (the number of particles of 10 μm and above ≤ 6000 particles, the number of particles of 25 μm and above ≤ 600 particles), the insoluble particles of all candidate prescriptions showed no obvious abnormalities during the entire inspection period and met the quality standards, among which F2-4-2 performed slightly better.

[0219] (5) DLS: There was no significant difference in particle size among all candidate formulations.

[0220] (6) CEX: There was no significant difference in the main peaks of charge isomers among all candidate formulations.

[0221] (7) SE-HPLC: There was no significant difference in the monomer purity of all candidate formulations.

[0222] (8) CE-SDS (NR): There was no significant difference in protein purity among all candidate formulations.

[0223] (9) ELISA-Binding: There was no significant difference in the binding activity of all candidate formulations.

[0224] Summary: A comprehensive evaluation of the three candidate formulations across all test parameters revealed no significant differences in protein stability between 0.02% (w / v) and 0.06% (w / v) PS80 concentrations. Therefore, we continued to use the 0.04% (w / v) PS80 concentration used in the excipient screening study and selected formulation F2-4-2 for subsequent formulation confirmation stability studies.

[0225] Example 5 Prescription Confirmation Stability

[0226] 5.1 Research Plan

[0227] (1) The formulation selected was 20 mM acetic acid-sodium acetate, pH 5.0; 8% (w / v) sucrose; and 0.04% (w / v) PS80 for formulation confirmation stability studies. (2) The packaging material selected was a 10 mL vial with a 10 mm rubber stopper and a 10 mm aluminum-plastic cap. (3) Specification: 50 mg / mL PVRIG / TIGIT dual antibody LC-BsAb-002; volume: 6 mL / 300 mg. Evaluation criteria included appearance, pH, protein concentration, dynamic light scattering (DLS), CEX, SE-HPLC, CE-SDS (NR&R), and binding activity (ELISA-Binding). The evaluation plan is detailed in Table 46.

[0228] Table 46 Stability test plan for prescription confirmation Note: X = appearance, pH, protein concentration, DLS, iCIEF, SE-HPLC, CE-SDS (NR), ELISA-Binding, insoluble particulate matter (MFI).

[0229] 5.2 Investigation Results

[0230] The main results of the formulation stability confirmation are summarized in Tables 47 and 48 below:

[0231] Table 47 Prescription confirmation stability results

[0232] Table 48 Prescription confirmation stability results

[0233] 5.3 Results Analysis

[0234] (1) Appearance: The appearance of the prescription did not change significantly during the entire investigation period.

[0235] (2) pH value: The pH of the formulation did not change significantly during the entire investigation period.

[0236] (3) Protein concentration: The protein concentration of this formulation did not change significantly during the entire observation period.

[0237] (4) Insoluble particles: The insoluble particles of this prescription showed no obvious abnormalities during the entire inspection period and met the quality standards.

[0238] (5) DLS: The particle size of this formulation did not change significantly during the entire observation period.

[0239] (6) CEX: When the sample was stored at low temperature, room temperature and high temperature, the change of the main peak of the charge isomer of the prescription met the quality standard.

[0240] (7) SE-HPLC: The purity of the monomers of this formulation is stable when the sample is at low temperature, room temperature and high temperature.

[0241] (8) CE-SDS (NR&R): When the sample is at low temperature, room temperature and high temperature, the protein purity of this formulation changes little and has good stability.

[0242] (9) ELISA-Binding: The binding activity of this formulation did not change significantly during the entire observation period.

[0243] Summary: All test indicators of this prescription meet the quality standards under the test conditions of 2-8℃, 25℃ and 40℃, and the stability is good. There is no significant difference between upright and inverted samples.

[0244] In summary, after pH / buffer system screening, excipient screening, surfactant strength screening, and formulation confirmation stability studies, the final formulation was determined to be 50 mg / mL PVRIG / TIGIT bispecific antibody; 20 mM acetic acid-sodium acetate, pH 5.0; 8% (w / v) sucrose; and 0.04% (w / v) PS80.

[0245] Although the present invention ultimately selects 20 mM acetic acid-sodium acetate, pH 5.0; 8% (w / v) sucrose; 0.04% (w / v) PS80 for the formulation, it does not mean that only this formulation can achieve the effect of stabilizing antibodies. As shown in the data of the aforementioned examples, the use of a histidine buffer system, as well as sorbitol and trehalose as excipients, can also achieve stable storage of antibodies.

Claims

1. A pharmaceutical composition comprising: (i) a bispecific antibody or an antigen-binding fragment thereof, comprising binding domains that specifically bind to PVRIG and TIGIT, (ii) buffer, (iii) non-reducing sugars, and (iv) nonionic surfactants, in, The bispecific antibody or antigen-binding fragment thereof comprises: (a) A first antigen-binding portion comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL form an anti-TIGIT antigen-binding domain; wherein the TIGIT VH comprises HCDR1, HCDR2, and HCDR3 represented by the following sequence: (1) HCDR1, HCDR2, and HCDR3 are SEQ ID NOs: 21, 22, and 23, respectively; or (2) HCDR1, HCDR2, and HCDR3 are SEQ ID NOs: 24, 25, and 26, respectively; or (3) a sequence having at least 90% identity with the sequence shown in (1) to (2) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions; The TIGIT VL comprises LCDR1, LCDR2, and LCDR3 shown in the following sequence: (1) LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 27, 28, and 29, respectively; or (2) LCDR1, LCDR2, and LCDR3 are SEQ ID NOs: 30, 31, and 32, respectively; or (3) a sequence having at least 90% identity with the sequence shown in (1) to (2) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions; (b) a second antigen-binding portion comprising a VHH that specifically binds to PVRIG, said VHH comprising CDR1, CDR2 and CDR3 as shown in the following sequence: (1) CDR1, CDR2, and CDR3 are SEQ ID NOs: 15, 16, and 17, respectively; or (2) CDR1, CDR2, and CDR3 are SEQ ID NOs: 18, 19, and 20, respectively; or (3) A sequence having at least 90% identity with the sequence shown in (1) to (2) above or having 1, 2, 3 or more amino acid insertions, deletions and / or substitutions, preferably, the substitutions are conservative amino acid substitutions.

2. The pharmaceutical composition according to claim 1, wherein The TIGIT VH comprises the amino acid sequence shown in any one of SEQ ID NO: 12 or 14; The TIGIT VL comprises the amino acid sequence shown in any one of SEQ ID NO: 11 or 13; The VHH that specifically binds to PVRIG comprises the amino acid sequence shown in any one of SEQ ID NO: 9 or 10.

3. The pharmaceutical composition according to claim 1 or 2, wherein The bispecific antibody or antigen-binding fragment thereof is LC-BsAb-002, LC-BsAb-006, LC-BsAb-009, or LC-BsAb-010; preferably LC-BsAb-002; The LC-BsAb-002 comprises: (1) LC with the amino acid sequence represented by SEQ ID NO: 1; and (2) HC with an amino acid sequence represented by SEQ ID NO: 2; The LC-BsAb-006 comprises: (3) LC with an amino acid sequence represented by SEQ ID NO: 3; and (4) HC with an amino acid sequence represented by SEQ ID NO: 4; The LC-BsAb-009 comprises: (1) LC with the amino acid sequence represented by SEQ ID NO: 5; and (2) HC with an amino acid sequence represented by SEQ ID NO: 6; The LC-BsAb-010 comprises: (3) LC with the amino acid sequence represented by SEQ ID NO: 7; and (4) HC whose amino acid sequence is represented by SEQ ID NO:

8.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The concentration of the PVRIG / TIGIT bispecific antibody or its antigen-binding fragment is about 10 mg / ml to about 200 mg / ml, preferably, about 20 mg / ml to about 100 mg / ml, about 20 mg / ml to about 80 mg / ml, or about 30 mg / ml to about 60 mg / ml; most preferably, 50 mg / mL.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein The buffer is selected from acetic acid-sodium acetate buffer or histidine-histidine hydrochloride buffer. Preferably, the buffer concentration is about 10mM to about 80mM, about 15mM to about 70mM, about 20mM to about 60mM, or about 20mM to about 30mM; most preferably, it is 20mM acetic acid-sodium acetate buffer.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein The non-reducing sugar is selected from sucrose, sorbitol or trehalose. Preferably, the concentration of the non-reducing sugar is about 6% to about 9% (w / v), about 7% to about 9% (w / v), or about 7% to about 8% (w / v); most preferably, it is 8% w / v sucrose.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein The nonionic surfactant is polysorbate 80. Preferably, the concentration of the nonionic surfactant is about 0.01% to about 0.10% (w / v), or about 0.02% to about 0.06% (w / v); most preferably, it is 0.04% (w / v) polysorbate 80.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein The pharmaceutical composition is an injection, preferably a subcutaneous injection or an intravenous injection.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein The pharmaceutical composition comprises: (i) 10 mg / ml to 200 mg / ml of a PVRIG / TIGIT bispecific antibody or an antigen-binding fragment thereof; (ii) 10 mM to 100 mM acetic acid-sodium acetate buffer; (iii) 6% to 10% (w / v) sucrose; (iv) 0.01% to 0.10% (w / v) polysorbate 80.

10. The pharmaceutical composition of any one of claims 1-9, comprising 50 mg / mL of the PVRIG / TIGIT bispecific antibody or its antigen-binding fragment, 20 mM acetic acid-sodium acetate buffer, 8% w / v sucrose, and 0.02% (w / v) polysorbate 80.

11. The pharmaceutical composition of any one of claims 1-9, comprising 50 mg / mL of the PVRIG / TIGIT bispecific antibody or its antigen-binding fragment, 20 mM acetic acid-sodium acetate buffer, 8% w / v sucrose, and 0.04% (w / v) polysorbate 80.

12. The pharmaceutical composition of any one of claims 1-9, comprising 50 mg / mL of the PVRIG / TIGIT bispecific antibody or its antigen-binding fragment, 20 mM acetic acid-sodium acetate buffer, 8% w / v sucrose, and 0.06% (w / v) polysorbate 80.

13. The pharmaceutical composition of any one of claims 1-12, wherein the pH of the pharmaceutical composition is 4.5-5.

5.

14. The pharmaceutical composition of claim 13, wherein the pH of the pharmaceutical composition is 5.0-5.

2.

15. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition is stable at 2°C to 8°C for at least 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, or 6 months.

16. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition is stable at 25°C for at least 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 3 months, or 6 months.

17. The pharmaceutical composition of any one of claims 1-14, wherein the pharmaceutical composition is stable at 40°C for at least 1 week, 2 weeks, 3 weeks or 4 weeks.

18. Use of the pharmaceutical composition according to any one of claims 1 to 17 in the preparation of a medicament for treating cancer or an infectious disease; wherein the cancer is selected from solid tumors and blood tumors.

19. The use according to claim 18, wherein The pharmaceutical composition is used in combination with another therapeutic agent, radiotherapy or surgery; wherein the additional therapeutic agent is selected from chemotherapy, oncolytic drugs, cytotoxic agents, cytokines, immunostimulatory antibodies, immunomodulatory drugs, activators of costimulatory molecules, inhibitors of inhibitory molecules, vaccines or cellular immunotherapy.

20. A method for treating cancer or infectious diseases, comprising administering an effective amount of the pharmaceutical composition of any one of claims 1 to 17 to a patient in need thereof; wherein the cancer is selected from solid tumors and hematological tumors.

21. The pharmaceutical composition according to any one of claims 1 to 17, for use in treating cancer or infectious diseases; wherein the cancer is selected from solid tumors and blood tumors.