WO2024153182A1 - Use of anti-pvrig/anti-tigit bispecific antibody in treatment of malignant tumors - Google Patents

Use of anti-pvrig/anti-tigit bispecific antibody in treatment of malignant tumors Download PDF

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WO2024153182A1
WO2024153182A1 PCT/CN2024/073012 CN2024073012W WO2024153182A1 WO 2024153182 A1 WO2024153182 A1 WO 2024153182A1 CN 2024073012 W CN2024073012 W CN 2024073012W WO 2024153182 A1 WO2024153182 A1 WO 2024153182A1
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pvrig
tigit
antibody
dose
seq
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PCT/CN2024/073012
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French (fr)
Chinese (zh)
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李炯雁
田吉
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山东先声生物制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum

Definitions

  • the present invention relates to the field of cancer treatment, and in particular, to the use of anti-PVRIG/anti-TIGIT bispecific antibodies or antigen-binding fragments thereof in the treatment of malignant tumors.
  • Cancer is characterized by the uncontrolled growth of a subpopulation of cells. Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries, with more than 14 million new cancer cases diagnosed and more than 8 million cancer deaths each year. Cancer care therefore represents a significant and growing burden on society.
  • Lung cancer is a malignant lung tumor and the leading cause of cancer death worldwide. Lung cancer is the most common cause of cancer death in men and the second most common cause of cancer death in women, second only to breast cancer.
  • Non-small cell lung cancer is the most common type of lung cancer, accounting for about 85% of lung cancers. About 75% of patients are already in the middle or advanced stages when they are discovered, and the 5-year survival rate is very low. In the past two decades, stage III non-small cell lung cancer (NSCLC) has remained a clinical challenge, and the 5-year survival rate of patients is also very low despite the aggressive treatment regimen of concurrent radiotherapy and chemotherapy.
  • Hepatocellular carcinoma is one of the most common tumors in the world and is a type of primary liver cancer. HCC is most common in patients with chronic liver disease, such as cirrhosis caused by hepatitis B or C infection, and HCC is usually diagnosed in the late stages of the above diseases.
  • the survival time of patients with advanced liver cancer is often only six months to one and a half years.
  • the recurrence rate of liver cancer is high, and the tumor recurrence and metastasis rate within 5 years after surgical resection is as high as 40% to 70%.
  • Immune checkpoint inhibitors provide a new clinical treatment approach for the treatment of NSCLC and HCC.
  • Immune checkpoints are a class of immunosuppressive molecules. Their physiological function is to regulate the intensity and breadth of immune responses to avoid the occurrence of autoimmunity. Tumor cells often use the characteristics of immune checkpoints to evade the attack of immune cells.
  • PVRIG is expressed on cells of NK cells and T cells and has several similarities to other known immune checkpoints.
  • PVRIG binds to its ligand (PVRL2)
  • an inhibitory signal is triggered, which acts to attenuate 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 thus modulates the immune response of NK cells and T cells.
  • Utilizing PVRIG antibodies that block binding to PVRL2 is a therapeutic approach to enhance NK cells and T cells to kill cancer cells. Blocking antibodies that bind to PVRIG and block the binding of its ligand PVRL2 have been produced.
  • TIGIT is another immune checkpoint target of interest, and binding to its cognate ligand PVR has been shown to directly inhibit NK cell and T cell cytotoxicity 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. In addition to its direct effects on T and NK cells, TIGIT can also induce PVR-mediated signaling in dendritic cells or tumor cells, leading to increased production of anti-inflammatory cytokines such as IL10. Notable However, TIGIT expression is closely associated with the expression of another important co-inhibitory receptor, PD-1. TIGIT and PD-1 are co-expressed on many human and mouse tumor-infiltrating lymphocytes (TILs).
  • TILs tumor-infiltrating lymphocytes
  • TIGIT and PVRIG belong to the DNAM superfamily and have been shown to co-express in a variety of tumor-infiltrating lymphocytes to exert immunosuppressive effects.
  • tumor-infiltrating effector T cells that co-express TIGIT, PVRIG, and PD-1 are considered to be the most important group of effector T cells in the infiltrating T cell population. Therefore, bispecific antibodies that can simultaneously target PVRIG and TIGIT have potential synergistic effects and are an attractive treatment for single antibody therapy. Such bispecific antibodies will allow simultaneous targeting of two immune checkpoint receptors while having potential further synergistic effects with existing anti-PD-1/L-1 antibody therapies, playing an important role in providing new therapeutic approaches in cancer treatment.
  • the present invention is specially proposed.
  • the present invention provides a method for preventing or treating malignant tumors, wherein the method comprises administering an anti-PVRIG/anti-TIGIT antibody or a pharmaceutical composition thereof to a patient in need thereof, wherein the pharmaceutical composition comprises a physiologically acceptable carrier.
  • the dosage of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is 1 mg-800 mg, preferably 5 mg-800 mg, 10 mg-800 mg, 20 mg-800 mg, 30 mg-800 mg, 5 mg-700 mg, 10 mg-700 mg, 20 mg-700 mg, 30 mg-700 mg, 5 mg-600 mg, 10 mg-600 mg, 20 mg-600 mg, 30 mg-600 mg, 50 mg-600 mg, 100 mg-600 mg, 300 mg-600 mg, 10 mg-300 mg, 30 mg-300 mg, 100 mg-300 mg, 10 mg-100 mg, 30 mg-100 mg, or 10 mg-30 mg.
  • the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is administered at a dosage of about 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg.
  • the malignant tumor is non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), melanoma, human pharyngeal carcinoma or other metastatic or locally advanced solid tumors.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • melanoma human pharyngeal carcinoma or other metastatic or locally advanced solid tumors.
  • the malignant tumor patient is a NSCLC patient
  • the patient is an incurable advanced NSCLC patient confirmed by histology or cytology;
  • the patients are NSCLC patients whose disease progressed after receiving at least 1 but no more than 2 systemic anti-tumor therapies;
  • the patients are NSCLC patients who have failed at least one established standard anti-tumor targeted therapy, or are not suitable for current standard treatment according to the judgment of the researcher.
  • the malignant tumor patient is an HCC patient
  • the patient is a HCC patient confirmed by histology or cytology;
  • the patient is an HCC patient who has failed at least one anti-angiogenic therapy, but no more than two systemic anti-tumor therapies;
  • the liver cancer is stage B or C based on the Barcelona Clinic Liver Cancer (BCLC) staging system, and is not suitable for Combined with surgery or local treatment, or progressed after surgery and/or local treatment;
  • BCLC Barcelona Clinic Liver Cancer
  • the liver cancer has a Child-Pugh score ⁇ 7 and is free of hepatic encephalopathy.
  • the anti-PVRIG/anti-TIGIT antibody comprises:
  • a first antigen binding portion which includes 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 antigen binding domain comprises HCDR1, HCDR2 and HCDR3 of the VH described in SEQ ID NO: 5; and the TIGIT antigen binding domain comprises LCDR1, LCDR2 and LCDR3 of the VL described in SEQ ID NO: 4;
  • a second antigen binding portion which comprises a VHH that specifically binds to PVRIG, wherein the VHH comprises CDR1, CDR2 and CDR3 of the sequence described in SEQ ID NO: 3.
  • the first antigen binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequence:
  • the second antigen binding portion comprises CDR1, CDR2 and CDR3 of the following sequence:
  • 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: 5; 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: 4; 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: 3.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises about 50 mg/mL of anti-PVRIG/anti-TIGIT antibody, about 20 mM acetic acid-sodium acetate buffer, about 8% w/v sucrose, and about 0.02%-0.06% polysorbate 80;
  • the pharmaceutical composition is in liquid form or a lyophilized powder formulation.
  • the dosage form of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is 100-400 mg/bottle, preferably 100-300 mg/bottle, 150-350 mg/bottle, 200-350 mg/bottle, 200-300 mg/bottle or 250-300 mg/bottle.
  • the dosage form of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is about 100 mg/bottle, 150 mg/bottle, 180 mg/bottle, 200 mg/bottle, 250 mg/bottle, 300 mg/bottle, 350 mg/bottle or 400 mg/bottle; preferably, the dosage form of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is a single-dose dosage form, each dose comprising a single dose that can be administered to a patient.
  • the amount of antibody that is effectively administered is preferably an amount of an antibody that is effectively administered.
  • the anti-PVRIG/anti-TIGIT antibody or pharmaceutical composition thereof is administered approximately once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, preferably once a week.
  • the anti-PVRIG/anti-TIGIT antibody or pharmaceutical composition thereof is administered by intravenous drip, intravenous injection, oral administration, subcutaneous injection, intramuscular injection or intratumor injection, preferably, by intravenous infusion.
  • the administration cycle of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is one week, two weeks, three weeks, four weeks, two months, three months, four months, five months, half a year or longer.
  • the time of each administration cycle is the same or different, and the interval between each administration cycle is the same or different.
  • the patient shows complete response or partial response according to RECIST version 1.1.
  • compositions comprising A and B should be understood as the following technical solution: a composition consisting of A and B, and a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition”.
  • pharmaceutical composition refers to a preparation that exists in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to the subject to whom 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.
  • pharmaceutical composition and “preparation” are not mutually exclusive.
  • pharmaceutical composition herein includes antibodies, antibody-derived molecules, etc.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of cancer.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, 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 or complete), whether detectable or undetectable.
  • Subjects in need of treatment include those who already have a condition or disease, as well as those who are susceptible to a condition or disease or those for whom a condition or disease is to be prevented.
  • slow down, alleviate, weaken, alleviate, and relieve their meanings also include elimination, disappearance, non-occurrence, and the like.
  • patient refers to a human being receiving treatment for a particular disease or condition as described herein.
  • subjects and patients include mammals such as humans, primates (eg, monkeys), or non-primate mammals receiving treatment for a disease or condition.
  • tumor refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant tumors are included in this definition.
  • tumor or “neoplasm” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • the term "dose" is the amount of a drug that elicits a therapeutic effect. Unless otherwise indicated, the dose is related to the amount of the drug in free form. If the drug is in the form of a pharmaceutically acceptable salt, the amount of the drug is increased proportionally compared to the amount of the drug in free form. For example, the dose will be stated on the product packaging or in a product information sheet.
  • TIGIT T cell immunoreceptor having Ig and ITIM domains
  • TIGIT antigen Vstm3
  • WUCAM WUCAM
  • TIGIT T cell immunoreceptor having Ig and ITIM domains
  • TIGIT antigen Vstm3
  • WUCAM WUCAM
  • 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 variant, amino acid variant or isoform, 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 of PVRIG and PVLR2) are used to enhance T cell and/or NK cell activation and to treat diseases such as cancer and pathogen infection.
  • anti-PVRIG/anti-TIGIT antibody and "bispecific PVRIG/TIGIT antibody” and “anti-PVRIG/anti-TIGIT bispecific antibody” are used interchangeably herein, and the anti-PVRIG/anti-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.
  • binding herein means that an antigen-binding molecule (e.g., an antibody) typically specifically binds to an antigen and substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is typically reflected by an equilibrium dissociation constant (KD), wherein a lower KD indicates a higher affinity. Taking antibodies as an example, high affinity typically refers to a KD of about 1 ⁇ 10 -7 M or less, about 1 ⁇ 10 -8 M or less, about 1 ⁇ 10 -9 M or less, about 1 ⁇ 10 -10 M or less, 1 ⁇ 10 -11 M or less, or 1 ⁇ 10 -12 M or less.
  • KD equilibrium dissociation constant
  • KD KD/Ka, wherein Kd represents the dissociation rate and Ka represents the association rate.
  • the equilibrium dissociation constant KD can be measured by methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.
  • antigen binding molecule is used in the broadest sense herein to refer to a molecule that specifically binds to an antigen.
  • antigen binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetics refers to organic compounds or binding domains that are able to specifically bind to an antigen but are unrelated to the antibody structure, and 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.
  • DARPins ankyrin repeat proteins
  • antibody herein is used in the broadest sense and refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of the immunoglobulin heavy chain and/or sufficient sequence from the variable region of the immunoglobulin light chain to be able to specifically bind to an antigen.
  • Antibodies herein encompass various forms and various 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 contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers.
  • Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used to transplant CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, etc.
  • the “antibody” herein includes a typical "four-chain antibody”, which belongs to an immunoglobulin composed of two heavy chains (HC) and two light chains (LC);
  • the heavy chain refers to a polypeptide chain, which is composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N-terminal to C-terminal; and, when the full-length antibody is an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain;
  • the light chain is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminal to C-terminal;
  • the heavy chains and the heavy chains, and the heavy chains and the light chains are connected by disulfide bonds to form a "Y"-shaped structure.
  • immunoglobulins Due to the different amino acid compositions and arrangement orders of the constant regions of the heavy chains of immunoglobulins, their antigenicity is also different. Based on this, the "immunoglobulins" in this article can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain and ⁇ chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into ⁇ chains or ⁇ chains by different constant regions. Each of the five types of Ig can have ⁇ chains or ⁇ chains.
  • antibody in this article also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNARs) found in cartilaginous fish such as sharks.
  • HCAbs heavy-chain antibodies
  • camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos)
  • the term “heavy chain antibody” refers to an antibody lacking the light chain of a conventional antibody.
  • the term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • the term “nanoantibody” refers to the natural heavy chain antibody lacking light chain that exists in camels. Cloning its variable region can obtain a single domain antibody consisting of only the heavy chain variable region, also called VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH Very domain of heavy chain of heavy chain antibody
  • the terms “nanobody” and “single domain antibody” have the same meaning and can be used interchangeably. They refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, a heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • 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, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).
  • Antibody herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.
  • 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 generated during the production process of the preparation, such variants are usually 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 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 production of the antibody or antigen-binding molecule by any particular method.
  • 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 using transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.
  • the term "monospecific” as used herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.
  • multispecific herein refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens.
  • terms such as “bispecific”, “trispecific”, “tetraspecific” and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valent herein refers to the presence of a specified number of binding sites in an antibody/antigen binding molecule.
  • the terms “monovalent”, “divalent”, “tetravalent” and “hexavalent” refer to the presence of one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively.
  • Frull length antibody “intact antibody,” and “intact antibody” are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody.
  • Antigen binding fragment and “antibody fragment” are used interchangeably herein, and they do not have the entire structure of a complete antibody, but only contain a partial or partial variant of a complete antibody, and the partial or partial variant 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, diabody and single domain antibody.
  • Papain digestion of intact antibodies generates two identical antigen-binding fragments, called “Fab” fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab fragment herein refers to an antibody fragment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody.
  • Fab'-SH is a fragment in which the cysteine residues of the constant domains carry a free sulfur Pepsin treatment generates a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region.
  • Fd refers to an antibody consisting of a VH and CH1 domain.
  • Fv refers to an antibody fragment consisting of a single-arm VL and VH domain.
  • the Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to the antibody. However, even a single variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • GGGGS linker having the amino acid sequence
  • Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).
  • diabody herein refers to an antibody whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • naked antibody herein refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term “conjugated antibody” refers to an antibody conjugated to a therapeutic agent or tracer, preferably, the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent and a photosensitizer.
  • variable region herein refers to the region of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen
  • variable domains of the heavy and light chains of natural antibodies generally have similar structures, each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., 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.
  • complementarity determining region and “CDR” are used interchangeably herein, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This part is also called the complementarity determining region because it can form precise complementarity with the antigen epitope in terms of spatial structure.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • the light chain variable region CDR can be abbreviated as LCDR.
  • framework region or “FR region” are interchangeable and refer to those amino acid residues in the heavy chain variable region or light chain variable region of the antibody except for the CDR.
  • a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR herein can be annotated and defined by methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, and the tool websites used include but are 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).
  • CDR herein includes overlaps and subsets of amino acid residues defined in different ways.
  • Kabat numbering system in this article generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • conservative amino acid generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity).
  • amino acids within each of the following groups are conservative amino acid residues of each other, and the replacement of the amino acid residues within the group is a replacement of conservative amino acids:
  • identity herein can be calculated in the following manner: to determine the percentage of "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 comparison 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, the molecules are identical at this position.
  • 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.
  • Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the percent identity between two amino acid sequences can be determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com) that has been integrated into the GAP program of the GCG software package, using 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. Score.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com) using the 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.
  • a particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • 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.
  • nucleic acid sequences and protein sequences described in the present invention can be further used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10.
  • gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See www.ncbi.nlm.nih.gov.
  • single-dose dosage form refers to a drug that contains an effective amount of drug for a single administration to a subject or patient.
  • the amount of drug contained in a single-dose dosage form can be converted by an effective dose.
  • the effective amount calculated in mg/ m2 can be converted into the drug content of each dose of drug in a single-dose dosage form based on the body surface area of a person.
  • the effective amount calculated in mg/kg can be converted into the drug content of each dose of drug in a single-dose dosage form based on the body weight of a person.
  • a fixed dose can also be used, that is, a certain dose of drug is given to the patient at one time regardless of body weight.
  • Fixed dose administration can be converted into the drug content of each dose of drug in a single-dose dosage form, for example, a fixed dose of 150 mg, which can be the drug content of each dose of drug in a single-dose dosage form.
  • the dosage form can be an injection, tablet, adhesive, suspension, aerosol, granule, capsule, ointment or suppository, etc.
  • the anti-TIGIT/anti-PVRIG antibody described herein can be administered in the form of a fixed dose.
  • G4S linker peptide herein refers to a GS combination of glycine (G) and serine (S), which is used to link multiple proteins together to form a fusion protein.
  • G glycine
  • S serine
  • a commonly used GS combination is (GGGGS)n, and the length of the linker sequence is changed by changing the size of n.
  • glycine and serine can also produce different linker sequences through other combinations, such as the (G4S)4Linker used in the present invention, the GS combination is GGGGS.
  • AE adverse effect
  • a medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity.
  • OS all survival
  • PFS progression Free Survival
  • object response rate in this article refers to the proportion of patients whose tumors shrink to a certain level and remain shrinking for a certain period of time, including cases of CR and PR.
  • the solid tumor response evaluation criteria (RECIST 1.1 standard) are used to assess the objective response of tumors. Subjects must have measurable tumor lesions at baseline, and the efficacy evaluation criteria are divided into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to the RECIST 1.1 standard.
  • DCR Disease Control Rate
  • partial remission in this article is: the sum of the target lesion diameters is reduced by at least 30% compared with the baseline level.
  • PD progressive disease
  • stable disease in this article means that the reduction of target lesions has not reached the level of PR, and the increase has not reached the level of PD, but is somewhere in between.
  • the minimum value of the sum of diameters can be used as a reference for research.
  • immunotherapy refers to treating a subject who has a disease or is at risk of infection or recurrence of a disease by methods including inducing, enhancing, suppressing or otherwise modifying an immune response.
  • Treatment or “therapy” of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the purpose of reversing, alleviating, improving, slowing down or preventing the onset, progression, severity or recurrence of symptoms, complications or conditions, or biochemical indicators associated with the disease.
  • administration method and “administration regimen” are used interchangeably to refer to the dosage and timing of each therapeutic agent in the combination of the present invention.
  • RECIST 1.1 efficacy criteria refers to the definition described by Eisenhauver et al., E.A. et al., Eur. J Cancer 45:228-247 (2009) for target lesions or non-target lesions based on the context of the measured response. Prior to immunotherapy, it was the most commonly used criterion for efficacy assessment in solid tumors.
  • ECOG scoring standard in this article is an indicator of the patient's general health status and tolerance to treatment based on his or her physical strength.
  • the ECOG physical status scoring standard is scored as follows: 0, 1, 2, 3, 4, and 5.
  • a score of 0 means that the ability to move is completely normal, with no difference from the ability to move before the onset of the disease.
  • a score of 1 means that the patient can move freely and engage in light physical activities, including general housework or office work, but cannot engage in heavier physical activities.
  • Figure 2 Changes in body weight of mice in each test group after administration in the humanized PBMC-B-NDG mouse Detroit 562 cell transplant tumor model
  • FIG. 3 Tumor growth curves of each test group in the humanized PBMC-B-NDG mouse A375 cell transplant tumor model.
  • G1 PBS;
  • G2 10mg/kg Atezolizumab;
  • G3 2.57mg/kg TIGIT-002-H4L3;
  • G4 1.36mg/kg PVRIG-50H1b;
  • G5 8.57mg/kg TIGIT-002-H4L3;
  • G6 4.53mg/kg PVRIG-50H1b;
  • G7 3mg/kg LC-BsAb-002;
  • G8 10mg/kg LC-BsAb-002
  • Figure 4 Changes in body weight of mice in each test group after administration in the humanized PBMC-B-NDG mouse A375 cell transplant tumor model.
  • G1 PBS;
  • G2 10mg/kg Atezolizumab;
  • G3 2.57mg/kg TIGIT-002-H4L3;
  • G4 1.36mg/kg PVRIG-50H1b;
  • G5 8.57mg/kg TIGIT-002-H4L3;
  • G6 4.53mg/kg PVRIG-50H1b;
  • G7 3mg/kg LC-BsAb-002;
  • G8 10mg/kg LC-BsAb-002
  • the anti-PVRIG humanized VHH antibody PVRIG-A50-H1b was connected to the N-terminus of the heavy chain of the anti-TIGIT humanized monoclonal antibody TIGIT-002-H4L3 using a G4S connecting peptide to generate an anti-PVRIG ⁇ TIGIT humanized bispecific antibody named LC-BsAb-002.
  • Table 1 shows the sequences of the heavy chain fusion polypeptide (HC) and light chain polypeptide (LC) of LC-BsAb-002
  • Table 2 shows the variable region sequence of LC-BsAb-002
  • Table 3 shows the CDR sequence of LC-BsAb-002 divided according to Kabat.
  • the anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 gene was transfected into CHO-K1 cells using genetic engineering technology, and clones with higher yields were selected through MSX pressure screening and subcultured in CD CHO medium.
  • Anti-TIGIT/anti-PVRIG antibodies were expressed using a fed-batch culture method.
  • the basal culture medium was Opma's CHO CDP9, the culture cycle was 14-16 days, the reactor control parameters were pH 6.6-7.2, dissolved oxygen (DO) was ⁇ 20%, the speed was 75RPM-80RPM, and the initial culture temperature was 36.0°C-37.0°C; when the culture reached the 6th day, the culture temperature was lowered to 33.5°C-34.5°C until harvest.
  • the feed media were XF04 and CD FS08 from Opma, which were fed every other day starting from Day 3, with feed volumes of 5% and 0.5% of the volume respectively, until harvest.
  • Purification of anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 uses multi-step chromatography, concentration and filtration unit operations to purify the antibody in sequence.
  • the supernatant harvest is captured by Protein A affinity chromatography (AT Protein A Diamond Plus).
  • the captured antibody solution is treated with low pH incubation to inactivate potential viruses. After neutralization of the antibody solution, the precipitate is removed by deep filtration.
  • anion exchange chromatography (Diamond Q) is performed to remove impurities such as HCD, HCP, and shed Protein A
  • cation exchange (Diamond S) chromatography is performed to remove impurities such as HCP and aggregates. Filtration is performed through a nanofiltration membrane package to remove potential exogenous and exogenous viruses.
  • the antibody solution is then concentrated and buffer exchanged using an ultrafiltration membrane package to complete the purification and obtain the anti-TIGIT/anti-PVRIG antibody protein stock solution.
  • the packaging material was 10mL vials, 10mm rubber stoppers and 10mm aluminum-plastic caps. Specification: 50mg/mL anti-PVRIG/anti-TIGIT antibody LC-BsAb-002; Filling volume: 6mL/300mg.
  • the inspection indicators include: appearance, pH, protein concentration, dynamic light scattering (DLS), cation exchange chromatography (CEX), purity (molecular exclusion chromatography (SE-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS (NR&R)) and binding activity (ELISA-Binding).
  • the inspection plan is detailed in Table 4.
  • X appearance, pH, protein concentration, DLS, iCIEF, SE-HPLC, CE-SDS (NR), ELISA-Binding, insoluble particles (MFI).
  • the final prescription was determined to be 50 mg/mL PVRIG/TIGIT bispecific antibody; 20 mM acetic acid-sodium acetate, pH 5.0; 8% (w/v) sucrose; 0.04% (w/v) PS80.
  • Example 2 Efficacy of anti-TIGIT/anti-PVRIG antibodies: Treatment in a humanized PBMC-B-NDG mouse Detroit 562 cell xenograft tumor model
  • 0.2 mL of 1640 medium containing 5 ⁇ 10 6 human PBMCs was inoculated intravenously into each mouse.
  • Three days after PBMC inoculation 0.1 mL of PBS containing 5 ⁇ 10 6 Detroit 562 human pharyngeal carcinoma cells was inoculated subcutaneously in the right axilla of each mouse.
  • the average tumor volume reached approximately 84.38 mm 3
  • 36 mice with medium tumor volume and medium PBMC humanization level were selected for enrollment.
  • mice were assigned to 6 experimental groups: PBS group, Atezolizumab 8.27 mg/kg group, LC-BsAb-002 1, 3, 10 and 30 mg/kg groups; administration began on the day of grouping by intraperitoneal injection, which was recorded as day 0, and was administered twice a week, i.e., once on days 0, 3, 7, 10, 14, and 17.
  • Figure 1 and Table 5 show that on the 17th day after grouping, the average tumor volume of the PBS group (G1) was 920.53 mm 3 .
  • the TGI of the Atezolizumab 8.27 mg/kg group was 24.63%; the TGI of the LC-BsAb-002 30, 10, 3, and 1 mg/kg groups were 68.10%, 60.07%, 63.21%, and 62.32%, respectively, which were significantly better than Atezolizumab 8.27 mg/kg (P ⁇ 0.05).
  • Relative tumor growth rate T/C% TRTV/CRTV ⁇ 100% (TRTV: average RTV of the drug administration group; CRTV: average RTV of the control group).
  • Relative tumor volume, RTV Vt/V0 (V0: tumor volume on day 0, Vt: tumor volume on day 17).
  • c.TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%.
  • Figure 2 and Table 6 show that the average body weight changes in the PBS group, Atezolizumab 8.27 mg/kg group, LC-BsAb-002 30, 10, 3, and 1 mg/kg groups were 4.0%, -3.6%, -9.8%, -1.3%, -3.8%, and -8.3%, respectively, compared with day 0.
  • weight loss was observed in the LC-BsAb-002-administered groups, the state and behavior of the mice were normal, indicating that the mice could tolerate LC-BsAb-002 at doses of 1 mg/kg to 30 mg/kg.
  • LC-BsAb-002 showed significant antitumor efficacy in the humanized PBMC-B-NDG mouse Detroit 562 cell transplant tumor model, and the TGI of the 30, 10, 3 and 1 mg/kg groups were 68.10%, 60.07%, 63.21% and 62.32%, respectively, which were significantly better than 8.27 mg/kg of Atezolizumab.
  • no animals stopped taking the drug.
  • weight loss was observed in the LC-BsAb-002-treated group, the mice were in normal condition and behavior, indicating that mice can tolerate 1 mg/kg to 30 mg/kg of LC-BsAb-002.
  • Example 3 Efficacy of anti-TIGIT/anti-PVRIG antibodies: Treatment in a humanized PBMC-B-NDG mouse A375 cell xenograft model
  • 0.1 mL of PBS containing 5 ⁇ 10 6 A375 human melanoma cells was subcutaneously inoculated in the right axilla of each mouse to form a tumor.
  • each mouse was intravenously inoculated with 0.2 mL of 1640 culture medium containing 5 ⁇ 10 6 human PBMCs.
  • the average tumor volume reached about 119.07 mm 3
  • 64 mice with medium tumor volume and medium PBMC humanization level greater than 0.8% were selected for enrollment.
  • mice were assigned to 8 experimental groups: PBS group, Atezolizumab 10 mg/kg group, TIGIT-002-H4L3 2.57 mg/kg group, PVRIG-50H1b 1.36 mg/kg group, TIGIT-002-H4L3 8.57 mg/kg group, PVRIG-50H1b 4.53 mg/kg group, LC-BsAb-002 3 mg/kg group and 10mg/kg group; drug administration began on the day of grouping, which was recorded as day 0, and was administered twice a week, i.e. once on days 0, 4, 8, 11, and 15.
  • the average tumor volume of the PBS group (G1) was 1153.11 mm 3 .
  • Relative tumor growth rate T/C% TRTV/CRTV ⁇ 100% (TRTV: average RTV of the drug administration group; CRTV: average RTV of the control group).
  • Relative tumor volume, RTV Vt/V0 (V0: tumor volume on day 0, Vt: tumor volume on day 17).
  • c.TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%.
  • LC-BsAb-002 showed significant anti-tumor efficacy in the humanized PBMC-B-NDG mouse A375 cell transplant tumor model, with a TGI of 47.02%, which was superior to equimolar anti-TIGIT, anti-PVRIG monotherapy and Atezolizumab.
  • TGI tumor growth factor
  • Example 4 A first-in-human, open-label, dose-escalation phase I study of safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of anti-TIGIT/anti-PVRIG antibodies in subjects with advanced solid tumors
  • This study aims to evaluate the safety and tolerability, pharmacokinetic/pharmacodynamic characteristics and preliminary efficacy of anti-TIGIT/anti-PVRIG antibody monotherapy in adult subjects with advanced solid tumors.
  • Part 1 includes dose escalation and dose escalation expansion parts.
  • Part 1 Dose Escalation: It is planned to enroll subjects with advanced solid tumors who have failed or are not suitable for standard treatment (SOC). A Bayesian Optimal Interval (BOIN) design will be used. Eligible subjects will start with a starting dose of 10 mg once a week (QW) and receive anti-TIGIT/anti-PVRIG antibody intravenous infusion (IV) on D1, D8, D15, and D22 of each cycle (28 days). The dose escalation plan is 10, 30, 100, 300, and 600 mg, using a semi-logarithmic escalation strategy, except for the last 2 dose groups, which use a 100% increase.
  • the target DLT incidence rate of MTD is set at 0.3. If the PK characteristics of a certain dose level are not fully investigated due to premature withdrawal of subjects, the investigator and the sponsor may jointly decide whether to enroll additional subjects at this dose level. The actual number of patients enrolled will depend on the dose level that reaches the MTD or maximum administered dose (MAD) and the additional enrollment of subjects in the completed cohort.
  • MAD maximum administered dose
  • the Safety Review Committee will decide whether to escalate the dose to the next dose level based on the overall data (including but not limited to safety, efficacy, PK and PD).
  • the dose escalation period in Part 1 based on safety, tolerability and pharmacokinetics, it may be allowed to explore intermediate dose levels or doses exceeding 600 mg QW jointly decided by the sponsor and the principal investigator (PI) through the SRC.
  • Other regimens may be considered based on the cumulative data of anti-TIGIT/anti-PVRIG antibodies.
  • the dose escalation expansion part will be decided based on the results of the dose escalation part. It is planned to enroll subjects with advanced solid tumors who have failed SOC or are not suitable for standard treatment. Additional subjects may be enrolled in the backfill dose cohort that has been demonstrated to not exceed the MTD or MAD and has been declared safe by the SRC to collect additional safety and PK data at this dose level.
  • subjects can continue to receive anti-TIGIT/anti-PVRIG antibody treatment until they meet the treatment discontinuation criteria.
  • additional subjects may be enrolled in Part 1 to determine safety and PK (e.g., specific dose cohorts may be enrolled if special requirements are met).
  • Part 2 will begin after the RD regimen of anti-TIGIT/anti-PVRIG antibody monotherapy is determined in Part 1, with the goal of better describing the safety and PK of anti-TIGIT/anti-PVRIG antibodies and evaluating the preliminary anti-tumor effects of the study drug.
  • the selection of the cohort expansion population will be based on the latest data, such as the results of Part 1 and the accumulated new data of other drugs with the same/similar mechanism of action, including but not limited to the following cancers: non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC) and other solid tumor groups.
  • NSCLC non-small cell lung cancer
  • HCC hepatocellular carcinoma
  • the SRC may limit the enrollment of the subgroup.
  • Eligible subjects will receive RD levels of anti-TIGIT/anti-PVRIG antibodies intravenously until treatment discontinuation criteria are met.
  • a Bayesian optimal interval (BOIN) design will be used to determine the MTD (if any) and RD. Only DLTs occurring within cycle 1 will be used for dose finding. As shown in Figure 5, the BOIN design uses the following rules optimized to minimize the probability of dose allocation errors to guide dose escalation/decrease:
  • the dose closest to the MTD is selected based on the ordinal regression specified in the Bayesian optimal interval (BOIN) design. Specifically, the dose whose ordinal estimate of the DLT rate is closest to the target DLT rate is selected. If there is a tie, the higher dose level is selected when the ordinal estimate is lower than the target DLT rate, and the lower dose level is selected when the ordinal estimate is greater than or equal to the target DLT rate.
  • BOIN Bayesian optimal interval
  • Grade 3 arthralgia that can be adequately managed with supportive care or resolves to Grade 2 within 7 days;
  • AEs that lead to discontinuation or delayed administration of LC-BsAb-002 for more than 7 days after review by the sponsor/SRC can also be considered as DLTs.
  • any clinically significant AE (e.g., late irAE) that occurs after the DLT assessment time window may be considered a DLT for subsequent dose level decisions.
  • the MTD was defined as the highest dose of LC-BsAb-002 showing a true toxicity rate ⁇ 0.3.
  • the MAD was defined as the highest dose of LC-BsAb-002 administered when the maximum tolerated dose (MTD) could not be determined.
  • the RD was selected by evaluating all available PK, PD, efficacy, safety, and tolerability data from Part 1. During the ramp-up process, if subjects at a specific dose (DLT has been evaluated) have a dosing-related efficacy response and/or PK and pharmacodynamic data indicate that drug exposure is within the estimated effective exposure range, expansion can be carried out at this dose level after discussion and approval by the SRC. PK sampling is also performed to further evaluate the safety, PK and PD characteristics and preliminary efficacy of this dose level, thereby helping to determine the appropriate recommended dose.
  • within-subject dose escalation is not recommended in this study, but in some special cases, within-subject dose escalation can be performed in eligible subjects based on the recommendations of the SRC.
  • the subject has tolerated treatment at a lower dose for at least four cycles as agreed by the attending investigator and the sponsor without experiencing any treatment-related toxicity prohibiting dose escalation (defined as CTCAE grade ⁇ 2 or worsening from baseline at the initially assigned lower dose).
  • the dose level and dosing regimen to be escalated must be a dose regimen for which DLT evaluation has been completed and has been determined to be safe for all subjects in the cohort and does not exceed the MTD.
  • ICF informed consent form
  • tumor tissue is not available despite best efforts, in certain circumstances, subjects may be enrolled after discussion with the sponsor or designee. (Please note that tumor samples will not be submitted until approval has been obtained from the relevant local health authorities.)
  • Dose escalation and dose expansion Subjects must have a histological or cytological diagnosis of any type of metastatic or locally advanced solid tumor that is not suitable for local treatment; subjects must have failed standard treatment or are not suitable for standard treatment.
  • CIT cancer immunotherapy
  • PD1/PDL1 inhibitors Subjects who have previously received cancer immunotherapy (CIT) including PD1/PDL1 inhibitors will be enrolled.
  • the selection of the cohort expansion population will be based on the latest data, such as the results of the Part 1 study and/or the accumulated new data of other drugs with the same/similar mechanism of action, including but not limited to the following tumor types:
  • ⁇ NSCLC cohort subjects with incurable advanced NSCLC confirmed by histology or cytology; subjects without driver gene mutations must have progressed after at least 1 but no more than 2 systemic anti-tumor therapies; subjects with driver gene mutations must have failed at least 1 established standard anti-tumor targeted therapy, or be unsuitable for current standard treatment based on the investigator's judgment. (Progression within 6 months after completion of adjuvant therapy is considered failure of first-line treatment.)
  • HCC confirmed by histology or cytology (excluding known fibrolamellar HCC or mixed cholangiocarcinoma and HCC); Child-Pugh score ⁇ 7, no hepatic encephalopathy; Barcelona Clinic Liver Cancer (BCLC) stage B or C, not suitable for surgery or local treatment, or progressing after surgery and/or local treatment; subjects who have failed at least 1 anti-angiogenic therapy but no more than 2 systemic anti-tumor therapies.
  • cytology excluding known fibrolamellar HCC or mixed cholangiocarcinoma and HCC
  • Child-Pugh score ⁇ 7, no hepatic encephalopathy
  • BCLC Barcelona Clinic Liver Cancer
  • ⁇ Other solid tumor cohort Subjects with histologically or cytologically confirmed malignant solid tumors other than NSCLC and HCC who have progressed after receiving at least 1 standard treatment.
  • Laboratory test values during the screening period must meet the following criteria (except for subjects who have received blood transfusion, blood component transfusion, or colony stimulating factors (e.g., granulocyte colony stimulating factor [G-CSF], granulocyte macrophage colony stimulating factor [GM-CSF], or recombinant erythropoietin, etc.) within 2 weeks before the first dose of study treatment):
  • colony stimulating factors e.g., granulocyte colony stimulating factor [G-CSF], granulocyte macrophage colony stimulating factor [GM-CSF], or recombinant erythropoietin, etc.
  • Hematology absolute neutrophil count (ANC) ⁇ 1.5 ⁇ 10 9/L; platelet ⁇ 100 ⁇ 10 9/L, hemoglobin ⁇ 90g/L.
  • UPN upper limit of normal
  • T-BIL serum total bilirubin
  • ⁇ Kidney Serum creatinine (Cr) ⁇ 1.5 ⁇ ULN, or Cr clearance ⁇ 60.0 mL/min (calculated using the Cockcroft-Gault formula or measured).
  • Body weight unit kg; serum Cr unit: mg/dL
  • Coagulation International normalized ratio (INR) ⁇ 1.5 or thromboplastin time (PT) ⁇ 1.5 ⁇ ULN (for subjects receiving anticoagulant therapy, PT and INR must be within the therapeutic range for the intended use of the anticoagulant).
  • LVEF left ventricular ejection fraction
  • WOCBP Women of childbearing potential
  • WOCBP must have a negative serum or urine pregnancy test result (minimum sensitivity of 25 IU/L or equivalent units of HCG) within 72 hours before the start of administration of the investigational drug.
  • Subjects have received cytotoxic therapy, anti-tumor targeted small molecules (such as tyrosine kinase inhibitors), or hormonal drugs within 5 half-lives or 2 weeks (whichever is shorter) before the first dose of study treatment; received monoclonal antibodies (mAb) within 5 half-lives or 4 weeks (the longer of the dose escalation part of Part 1 of the study; the shorter of the dose escalation extension of Part 1 and Part 2 of the study) before the first dose of study treatment; received anti-tumor Chinese medicine preparations or any radiotherapy within 2 weeks before the first dose of study treatment.
  • cytotoxic therapy such as tyrosine kinase inhibitors
  • mAb monoclonal antibodies
  • Subjects have not recovered from AEs caused by previous anti-tumor treatment (i.e., recovered to ⁇ Grade 1 or baseline level). Note: Subjects with ⁇ Grade 2 AEs that do not affect the safety of the subject are eligible to participate in the study, such as ⁇ Grade 2 alopecia and neuropathy caused by chemotherapy.
  • CNS metastases/leptomeningeal disease requiring immediate radiotherapy or steroid treatment (subjects with controlled CNS metastases may participate in this trial, provided that they are clinically stable for at least 4 weeks before entering the study, have no evidence of new brain metastases or brain metastases expansion, and have stopped taking steroids for at least 2 weeks before the first dose of study treatment);
  • Subjects with a history of active tuberculosis infection within 1 year Subjects with a history of active tuberculosis are suitable for inclusion if the investigator determines that there is no evidence of active tuberculosis more than 1 year before the first dose of study treatment;
  • Subjects with clinically significant cardiovascular disease within 6 months before the first administration of study treatment including but not limited to myocardial infarction, severe/unstable angina, primary cardiomyopathy, cerebrovascular accident (including transient ischemic attack, cerebral hemorrhage, cerebral infarction) or congestive heart failure (New York Heart Association functional grade>2); symptomatic coronary heart disease requiring drug treatment; arrhythmia requiring drug treatment; electrocardiogram showing QTcF interval>480ms; or uncontrolled hypertension (systolic blood pressure ⁇ 160mmHg and/or diastolic blood pressure ⁇ 100mmHg after adequate drug treatment);
  • steroids or adrenal replacement therapy with >10 mg prednisone or equivalent are permitted if there is no active autoimmune disease.
  • Screen failure is when a subject was assigned to the study but did not receive treatment. The following information should be provided for subjects who failed the screen:
  • Demographics including race/ethnicity, age, and gender as permitted by local regulations
  • the results closest to the estimated dosing date should be used for inclusion/exclusion criteria assessment.
  • the investigator should determine whether the subject can be rescreened after communicating with the sponsor's medical monitor.
  • the investigator should discuss with the sponsor and determine whether the subject can continue with the study treatment.
  • Anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 injection. Active ingredient: anti-TIGIT/anti-PVRIG antibody, 50 mg/mL. Excipients: acetic acid-sodium acetate, pH 5.0; sucrose; PS80.
  • Anti-TIGIT/anti-PVRIG antibodies are administered by intravenous infusion.
  • the dose escalation plan is 10, 30, 100, 300 and 600 mg, using a semi-logarithmic escalation strategy, except for the last 2 dose groups, which use 100% escalation.
  • Starting from the first cycle every 28 days is 1 cycle, and QW administration is continued until disease progression, intolerable toxicity, withdrawal of informed consent or death.
  • the first infusion of all subjects should be completed in no less than 90 minutes. Adjust the infusion rate according to infusion-related operations to ensure the safety of the subjects. If no infusion reaction is observed, the subsequent infusion time shall be no less than 60 minutes.
  • dose levels not exceeding the MTD such as intermediate dose levels or doses above 600 mg QW or alternative dosing regimens may be evaluated based on safety, tolerability, and pharmacokinetics or as jointly determined by the sponsor and PI through the SRC during Part 1.
  • Subjects will continue to receive study treatment until intolerable toxicity occurs, or until the investigator determines that the subject lacks clinical benefit after comprehensive evaluation of imaging and laboratory test data and the subject's clinical condition (e.g., worsening of tumor symptoms), or until the subject voluntarily withdraws from study treatment, whichever occurs first.
  • LC-BsAb-002 The dose of LC-BsAb-002 will not be reduced unless the subject is likely to benefit from treatment as assessed by the investigator and approved by the sponsor.
  • TEAEs treatment-emergent adverse events
  • LC-BsAb-002 administration may be restarted after the TEAE resolves to ⁇ Grade 1 or baseline value (or Grade 2, if the investigator determines that there is no safety risk to the subject).
  • LC-BsAb-002 should be permanently discontinued unless otherwise discussed with the sponsor.
  • the dosing window for the second cycle of treatment is ⁇ 3 days. LC-BsAb-002 needs to be administered during the window period. If the dosing window is missed, LC-BsAb-002 will skip this administration and be administered at the next scheduled visit.
  • LC-BsAb-002 should be permanently discontinued.
  • LC-BsAb-002 dosing may be interrupted for circumstances other than treatment-related AEs, such as medical/surgical events unrelated to study treatment or administrative reasons. In such cases, subjects should be restarted on study treatment within 4 weeks of the last dose unless otherwise discussed with the sponsor. The reason for interruption should be recorded in the patient's study record.
  • the NCI-CTCAE v5.0 standard will be used for safety evaluation.
  • ⁇ Hematological examination WBC, ANC, LYM, MONO, EOS, BASO, RBC, HB, PLT.
  • ⁇ Urinalysis pH, UWBC, urine protein, URBC.
  • ⁇ Blood biochemistry TBIL, DBIL, ALT, AST, ALP, GGT, TP, ALB, urea or BUN, Cr, UA, Glu, K, Na, Cl, Ca, Mg, P, LIP, AMY, LDL, HDL, LDH.
  • Cardiac enzymes CK, CK-MB; cTnT or cTnI (if necessary).
  • ⁇ Coagulation INR or PT.
  • ⁇ Thyroid function tests FT3 or T3, FT4 or T4, TSH.
  • HIV HBsAg, HBsAb, HBcAb, HCV-Ab; quantitative HCV-RNA and quantitative HBV-DNA (if necessary).
  • the investigator should conduct a comprehensive physical examination of the subject, including height, weight, general condition, skin/mucous membranes, facial features, head and neck, lungs/chest, abdomen, spine/limbs, nervous system, and urogenital system. Height only needs to be measured during the screening period.
  • the investigator may conduct specific physical examinations based on symptoms.
  • the investigator or other researchers authorized by the investigator should determine the subject's vital signs according to the protocol.
  • Vital signs include body temperature, heart rate, respiratory rate and blood pressure.
  • a standard 12-lead ECG including measurement of heart rate and QTcF interval, should be performed at the site using standard protocols. The timing of ECG measurements is specified in the evaluation schedule. Additional ECGs may be performed as clinically indicated.
  • Subjects will undergo an echocardiogram during the screening period and LVEF will be recorded. Additional echocardiograms may be performed at the discretion of the investigator based on ECG results during study treatment.
  • Serum samples will be collected for determination of the pharmacokinetics and immunogenicity of LC-BsAb-002.
  • Blood samples will be collected by direct venipuncture or by insertion of an indwelling cannula in the forearm vein, with samples collected from the arm contralateral to the infusion site, with 4 mL of venous blood collected each time for PK testing and 4.5 mL of venous blood collected for immunogenicity testing (3 mL for ADA testing and backup, 1.5 mL for Nab testing).
  • PK and immunogenicity ((ADA/Nab)) samples the exact date and time of administration, as well as the actual date and time of blood sample collection, must be recorded in the appropriate eCRF. Please refer to the central laboratory manual for detailed information on blood sample collection, handling, storage, and transportation.
  • the PK and immunogenicity (ADA/Nab) sampling strategy of Part 2 can be adjusted and optimized based on the actual measured results of the clinical pharmacokinetic characteristics of Part 1 or changes in the medication regimen.
  • Enhanced CT or MRI should be used to obtain tumor images.
  • the scan area should include the chest, abdomen, pelvis and brain. Whether other areas need to be scanned will be determined by the investigator based on specific circumstances.
  • the imaging method for the same subject should be consistent throughout the trial (including imaging method, range, mode, contrast agent, etc.).
  • Baseline imaging assessment of the tumor will be performed 4 weeks before the first dose. If the tumor assessment is performed before the signing of the informed consent form and is performed within 4 weeks before the first dose, this result can be used in place of the baseline imaging assessment with the consent of the sponsor. At screening, the investigator will confirm the target lesions and baseline imaging according to the RECIST version 1.1 criteria.
  • Imaging assessments will be performed every 8 ⁇ 1 weeks after the first dose. Investigators may perform unplanned imaging assessments based on the actual situation of the subjects. The timing of imaging assessments should follow calendar days and should not be changed due to treatment delays or unplanned imaging assessments. Imaging assessments should continue until the start of new anti-tumor treatment, disease progression, withdrawal from the study, or death, whichever occurs first. If an unplanned imaging assessment is performed within 4 weeks of the next scheduled imaging examination time point, the next scheduled imaging assessment may not be performed, and imaging assessments may continue at subsequent scheduled imaging time points.
  • tumor efficacy evaluation is divided into CR (complete remission), PR (partial remission), SD (stable disease) and PD (progressive disease).
  • CR complete remission
  • PR partial remission
  • SD stable disease
  • PD progressive disease
  • CR or PR remission should be confirmed ⁇ 4 weeks after the first recorded date of PR or CR.
  • PD progressive disease
  • treatment may continue until the investigator determines that the subject lacks clinical benefit after a comprehensive evaluation of the imaging and laboratory test data and the subject's clinical condition (e.g., worsening of tumor symptoms), or until the subject voluntarily withdraws from the study treatment (whichever occurs first).
  • Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). All AEs occurring during the study will be included in a list and summarized by MedDRA system organ class and preferred term. Additional safety summaries of clinical laboratory tests, vital signs, ECOG performance status score, ECG, etc. will be provided.
  • MedDRA Medical Dictionary for Regulatory Activities
  • Descriptive statistical methods were used to analyze efficacy indicators, including ORR, DCR, PFS, and OS.
  • the primary efficacy analysis will be performed on the confirmed best overall response ORR approximately 16 weeks after the last subject received the first infusion or at the end of the study (whichever occurs first).
  • the Kaplan-Meier method was used to summarize DOR, PFS, and OS.

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Abstract

A use of an anti-PVRIG/anti-TIGIT bispecific antibody or a pharmaceutical composition comprising same in the treatment of malignant tumors such as non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC) or other metastatic or locally advanced solid tumors.

Description

抗PVRIG/抗TIGIT双特异性抗体在治疗恶性肿瘤中的用途Use of anti-PVRIG/anti-TIGIT bispecific antibodies in the treatment of malignant tumors 技术领域Technical Field
本发明涉及癌症治疗领域，具体而言，涉及抗PVRIG/抗TIGIT双特异性抗体或其抗原结合片段在治疗恶性肿瘤中的用途。The present invention relates to the field of cancer treatment, and in particular, to the use of anti-PVRIG/anti-TIGIT bispecific antibodies or antigen-binding fragments thereof in the treatment of malignant tumors.
背景技术Background technique
癌症的特征是细胞亚群不受控制的生长。癌症是发达国家的主要死因，也是发展中国家的第二大死因，每年新诊断的癌症病例超过1400万，癌症死亡人数超过800万。因此，癌症护理代表了一项重大且不断增加的社会负担。Cancer is characterized by the uncontrolled growth of a subpopulation of cells. Cancer is the leading cause of death in developed countries and the second leading cause of death in developing countries, with more than 14 million new cancer cases diagnosed and more than 8 million cancer deaths each year. Cancer care therefore represents a significant and growing burden on society.
肺癌是恶性的肺部肿瘤，是全球癌症死亡的主要原因。肺癌在男性中是常见的癌症致死病因，在女性则仅次于乳癌，列名第二。Lung cancer is a malignant lung tumor and the leading cause of cancer death worldwide. Lung cancer is the most common cause of cancer death in men and the second most common cause of cancer death in women, second only to breast cancer.
非小细胞肺癌(NSCLC)是最常见的肺癌种类，约占肺癌的85％。约75％的患者发现时已处于中晚期，5年生存率很低。近二十年来，III期非小细胞肺癌(NSCLC)在临床上仍是一项具有挑战性的工作，尽管在同期进行放化疗的激进治疗方案下，患者的5年生存率也很低。Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 85% of lung cancers. About 75% of patients are already in the middle or advanced stages when they are discovered, and the 5-year survival rate is very low. In the past two decades, stage III non-small cell lung cancer (NSCLC) has remained a clinical challenge, and the 5-year survival rate of patients is also very low despite the aggressive treatment regimen of concurrent radiotherapy and chemotherapy.
肝细胞癌(HCC)是全球最常见的肿瘤之一，是一种原发性肝癌类型。肝细胞癌最常见于慢性肝病患者，如乙肝或丙肝感染引起的肝硬化，HCC通常在上述疾病的病程晚期诊断。晚期肝癌患者的生存时间往往只有半年到一年半。此外，肝癌的复发率较高，手术切除后的5年肿瘤复发转移率高达40％～70％。Hepatocellular carcinoma (HCC) is one of the most common tumors in the world and is a type of primary liver cancer. HCC is most common in patients with chronic liver disease, such as cirrhosis caused by hepatitis B or C infection, and HCC is usually diagnosed in the late stages of the above diseases. The survival time of patients with advanced liver cancer is often only six months to one and a half years. In addition, the recurrence rate of liver cancer is high, and the tumor recurrence and metastasis rate within 5 years after surgical resection is as high as 40% to 70%.
因此，该领域对于开发用于治疗肺癌例如非小细胞肺癌、肝癌的有效免疫疗法存在未满足的需求。Therefore, there is an unmet need in the field for developing effective immunotherapies for treating lung cancer, such as non-small cell lung cancer, and liver cancer.
近年来，通过抑制免疫检查点来激活人体自身免疫系统，使其发挥攻击肿瘤细胞作用的相关研究进展迅速，免疫检查点抑制剂为治疗NSCLC和HCC，提供了新的临床治疗途径。免疫检查点是一类免疫抑制性的分子。它们的生理学功能是调节免疫反应的强度和广度，从而避免自身免疫的发生。而肿瘤细胞往往利用免疫检查点的特性来逃避免疫细胞的攻击。In recent years, research on activating the body's own immune system by inhibiting immune checkpoints to enable it to attack tumor cells has progressed rapidly. Immune checkpoint inhibitors provide a new clinical treatment approach for the treatment of NSCLC and HCC. Immune checkpoints are a class of immunosuppressive molecules. Their physiological function is to regulate the intensity and breadth of immune responses to avoid the occurrence of autoimmunity. Tumor cells often use the characteristics of immune checkpoints to evade the attack of immune cells.
PVRIG在NK细胞和T细胞的细胞上表达，并且与其它已知的免疫检查点具有若干相似之处。当PVRIG与其配体(PVRL2)结合时，引发抑制信号，其起到减弱NK细胞和T细胞针对靶细胞的免疫反应(即类似于PD-1/PD-L1)的作用。阻断PVRL2与PVRIG的结合会切断PVRIG的这种抑制信号，并因此调节NK细胞和T细胞的免疫反应。利用阻断与PVRL2结合的PVRIG抗体是一种增强NK细胞和T细胞杀伤癌细胞的治疗方法。已经产生了结合PVRIG并阻断其配体PVRL2的结合的阻断抗体。PVRIG is expressed on cells of NK cells and T cells and has several similarities to other known immune checkpoints. When PVRIG binds to its ligand (PVRL2), an inhibitory signal is triggered, which acts to attenuate 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 thus modulates the immune response of NK cells and T cells. Utilizing PVRIG antibodies that block binding to PVRL2 is a therapeutic approach to enhance NK cells and T cells to kill cancer cells. Blocking antibodies that bind to PVRIG and block the binding of its ligand PVRL2 have been produced.
类似地，TIGIT是另一个所关注的免疫检查点标靶，已经证明与其同源配体PVR的结合通过其细胞内ITIM域直接抑制NK细胞和T细胞的细胞毒性。TIGIT基因的敲除或TIGIT/PVR相互作用的阻断抗体已展示在体外可以增强NK细胞杀伤，或者加剧体内自身免疫疾病。除了对T细胞和NK细胞的直接作用外，TIGIT还可在树突状细胞或肿瘤细胞中诱导PVR介导的信号传导，从而引起抑炎细胞因子(例如IL10)的产生增加。值得注意的 是，TIGIT表达与另一种重要的共抑制受体PD-1的表达密切相关。TIGIT和PD-1在许多人类和鼠类的肿瘤浸润淋巴细胞(TIL)上共表达。Similarly, TIGIT is another immune checkpoint target of interest, and binding to its cognate ligand PVR has been shown to directly inhibit NK cell and T cell cytotoxicity 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. In addition to its direct effects on T and NK cells, TIGIT can also induce PVR-mediated signaling in dendritic cells or tumor cells, leading to increased production of anti-inflammatory cytokines such as IL10. Notable However, TIGIT expression is closely associated with the expression of another important co-inhibitory receptor, PD-1. TIGIT and PD-1 are co-expressed on many human and mouse tumor-infiltrating lymphocytes (TILs).
TIGIT和PVRIG同属于DNAM超家族，并被证明在多种肿瘤浸润淋巴细胞中共表达发挥免疫抑制作用，同时，TIGIT、PVRIG和PD-1共表达的肿瘤浸润效应T细胞被认为是浸润T细胞群体中最主要的一群效应T细胞。因此，能够同时靶向PVRIG和TIGIT的双特异性抗体具有有潜在的协同效果，是用于单一抗体疗法的有吸引力的治疗方式。此类双特异性抗体将允许同时靶向两个免疫检查点受体同时能与现有的抗PD-1/L-1抗体疗法有潜在的进一步协同效果，在癌症治疗中提供新到的治疗手段发挥重要作用。TIGIT and PVRIG belong to the DNAM superfamily and have been shown to co-express in a variety of tumor-infiltrating lymphocytes to exert immunosuppressive effects. At the same time, tumor-infiltrating effector T cells that co-express TIGIT, PVRIG, and PD-1 are considered to be the most important group of effector T cells in the infiltrating T cell population. Therefore, bispecific antibodies that can simultaneously target PVRIG and TIGIT have potential synergistic effects and are an attractive treatment for single antibody therapy. Such bispecific antibodies will allow simultaneous targeting of two immune checkpoint receptors while having potential further synergistic effects with existing anti-PD-1/L-1 antibody therapies, playing an important role in providing new therapeutic approaches in cancer treatment.
发明内容Summary of the invention
鉴于双特异性抗体潜在的协同效果，以及免疫检查点抑制剂对于预防或治疗恶性肿瘤的良好前景，特提出本发明。In view of the potential synergistic effects of bispecific antibodies and the good prospects of immune checkpoint inhibitors for preventing or treating malignant tumors, the present invention is specially proposed.
本发明提供了一种预防或治疗恶性肿瘤的方法，其中，所述方法包括向有此需要的患者施用抗PVRIG/抗TIGIT抗体或其药物组合物，所述药物组合物包含生理上可接受的载体。The present invention provides a method for preventing or treating malignant tumors, wherein the method comprises administering an anti-PVRIG/anti-TIGIT antibody or a pharmaceutical composition thereof to a patient in need thereof, wherein the pharmaceutical composition comprises a physiologically acceptable carrier.
在一些实施例中，所述抗PVRIG/抗TIGIT抗体或其药物组合物的给药剂量为1mg-800mg，优选地，为5mg-800mg，10mg-800mg，20mg-800mg，30mg-800mg，5mg-700mg，10mg-700mg，20mg-700mg，30mg-700mg，5mg-600mg，10mg-600mg，20mg-600mg，30mg-600mg，50mg-600mg，100mg-600mg，300mg-600mg，10mg-300mg，30mg-300mg，100mg-300mg，10mg-100mg，30mg-100mg，或10mg-30mg。In some embodiments, the dosage of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is 1 mg-800 mg, preferably 5 mg-800 mg, 10 mg-800 mg, 20 mg-800 mg, 30 mg-800 mg, 5 mg-700 mg, 10 mg-700 mg, 20 mg-700 mg, 30 mg-700 mg, 5 mg-600 mg, 10 mg-600 mg, 20 mg-600 mg, 30 mg-600 mg, 50 mg-600 mg, 100 mg-600 mg, 300 mg-600 mg, 10 mg-300 mg, 30 mg-300 mg, 100 mg-300 mg, 10 mg-100 mg, 30 mg-100 mg, or 10 mg-30 mg.
更优选地，所述抗PVRIG/抗TIGIT抗体或其药物组合物的给药剂量为约1mg，5mg，10mg，15mg，20mg，30mg，40mg，50mg，60mg，70mg，80mg，90mg，100mg，150mg，200mg，250mg，300mg，350mg，400mg，450mg，500mg，550mg，600mg，650mg，700mg，750mg，或800mg。More preferably, the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is administered at a dosage of about 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg.
在一些实施例中，所述恶性肿瘤为非小细胞肺癌(NSCLC)，肝细胞癌(HCC)，黑色素瘤，人咽头癌或其他转移性或局部晚期实体瘤。In some embodiments, the malignant tumor is non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), melanoma, human pharyngeal carcinoma or other metastatic or locally advanced solid tumors.
在一些实施例中，所述恶性肿瘤患者为NSCLC患者；In some embodiments, the malignant tumor patient is a NSCLC patient;
优选地，所述患者为经组织学或细胞学证实的不可治愈的晚期NSCLC患者；Preferably, the patient is an incurable advanced NSCLC patient confirmed by histology or cytology;
优选地，对于无驱动基因突变的患者，所述患者为接受至少1种但不超过2种系统抗肿瘤治疗后疾病进展的NSCLC患者；Preferably, for patients without driver gene mutations, the patients are NSCLC patients whose disease progressed after receiving at least 1 but no more than 2 systemic anti-tumor therapies;
优选地，对于有驱动基因突变的患者，所述患者为接受至少1种已确定的标准抗肿瘤靶向治疗失败，或根据研究者的判断不适合目前的标准治疗的NSCLC患者。Preferably, for patients with driver gene mutations, the patients are NSCLC patients who have failed at least one established standard anti-tumor targeted therapy, or are not suitable for current standard treatment according to the judgment of the researcher.
在一些实施例中，所述恶性肿瘤患者为HCC患者；In some embodiments, the malignant tumor patient is an HCC patient;
优选地，所述患者为经组织学或细胞学证实的HCC患者；Preferably, the patient is a HCC patient confirmed by histology or cytology;
优选地，所述患者为经过至少1种含抗血管生成疗法的治疗失败，但不超过2种全身抗肿瘤治疗的HCC患者；Preferably, the patient is an HCC patient who has failed at least one anti-angiogenic therapy, but no more than two systemic anti-tumor therapies;
优选地，所述肝癌为基于巴塞罗那临床肝癌(BCLC)分期系统的B期或C期，不适 合手术或局部治疗，或在手术和/或局部治疗后进展；Preferably, the liver cancer is stage B or C based on the Barcelona Clinic Liver Cancer (BCLC) staging system, and is not suitable for Combined with surgery or local treatment, or progressed after surgery and/or local treatment;
优选地，所述肝癌为Child-Pugh评分≤7且无肝性脑病。Preferably, the liver cancer has a Child-Pugh score ≤ 7 and is free of hepatic encephalopathy.
在一些实施例中，所述抗PVRIG/抗TIGIT抗体包含：In some embodiments, the anti-PVRIG/anti-TIGIT antibody comprises:
(a)第一抗原结合部分，其包括重链可变区(VH)和轻链可变区(VL)，所述VH和VL形成抗TIGIT抗原结合域；其中，所述TIGIT抗原结合域包含SEQ ID NO：5所述VH的HCDR1、HCDR2和HCDR3；所述TIGIT抗原结合域包含SEQ ID NO：4所述VL的LCDR1、LCDR2和LCDR3；(a) a first antigen binding portion, which includes 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 antigen binding domain comprises HCDR1, HCDR2 and HCDR3 of the VH described in SEQ ID NO: 5; and the TIGIT antigen binding domain comprises LCDR1, LCDR2 and LCDR3 of the VL described in SEQ ID NO: 4;
(b)第二抗原结合部分，其包括特异性结合PVRIG的VHH，所述VHH包含SEQ ID NO：3所述序列的CDR1、CDR2和CDR3。(b) a second antigen binding portion, which comprises a VHH that specifically binds to PVRIG, wherein the VHH comprises CDR1, CDR2 and CDR3 of the sequence described in SEQ ID NO: 3.
优选地，所述第一抗原结合部分包含以下序列的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3：Preferably, the first antigen binding portion comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the following sequence:
(1)分别为SEQ ID NO：9、10、11、12、13和14；或(1) SEQ ID NO: 9, 10, 11, 12, 13 and 14 respectively; or
(2)与(1)所示序列具有至少90％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换；(2) a sequence having at least 90% identity with the sequence shown in (1) or having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions, preferably, the substitutions are conservative amino acid substitutions;
所述第二抗原结合部分包含以下序列的CDR1、CDR2和CDR3：The second antigen binding portion comprises CDR1, CDR2 and CDR3 of the following sequence:
(1)分别为SEQ ID NO：6、7和8；或(1) SEQ ID NO: 6, 7 and 8 respectively; or
(3)与(1)所示序列具有至少90％同一性或具有1、2、3或更多个氨基酸插入、缺失和/或替换的序列，优选地，所述替换为保守氨基酸的替换。(3) A sequence having at least 90% identity with the sequence shown in (1) or having 1, 2, 3 or more amino acid insertions, deletions and/or substitutions, preferably, the substitutions are conservative amino acid substitutions.
优选地，所述第一抗原结合部分的VH包含与SEQ ID NO：5所示氨基酸序列至少90％同一性的序列；所述第一抗原结合部分的VL包含与SEQ ID NO：4所示氨基酸序列至少90％同一性的序列；所述第二抗原结合部分包含与SEQ ID NO：3所示氨基酸序列至少90％同一性的序列。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: 5; 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: 4; 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: 3.
在一些实施例中，所述药物组合物包含：In some embodiments, the pharmaceutical composition comprises:
(i)约10mg/ml至约200mg/ml的抗PVRIG/抗TIGIT抗体；(i) about 10 mg/ml to about 200 mg/ml of an anti-PVRIG/anti-TIGIT antibody;
(ii)约10mM至约100mM的醋酸-醋酸钠缓冲液；(ii) about 10 mM to about 100 mM acetic acid-sodium acetate buffer;
(iii)约6％至约10％(w/v)蔗糖；和(iii) about 6% to about 10% (w/v) sucrose; and
(iv)约0.01％至约0.10％(w/v)聚山梨酯80；(iv) about 0.01% to about 0.10% (w/v) polysorbate 80;
优选地，所述药物组合物包含约50mg/mL的抗PVRIG/抗TIGIT抗体、约20mM醋酸-醋酸钠缓冲液、约8％w/v蔗糖、和约0.02％-0.06％聚山梨酯80；Preferably, the pharmaceutical composition comprises about 50 mg/mL of anti-PVRIG/anti-TIGIT antibody, about 20 mM acetic acid-sodium acetate buffer, about 8% w/v sucrose, and about 0.02%-0.06% polysorbate 80;
优选的，所述药物组合物为液体形式或冻干粉制剂。Preferably, the pharmaceutical composition is in liquid form or a lyophilized powder formulation.
在一些实施例中，所述抗PVRIG/抗TIGIT抗体或其药物组合物的剂型规格为100-400mg/瓶，优选100-300mg/瓶，150-350mg/瓶，200-350mg/瓶，200-300mg/瓶或250-300mg/瓶。In some embodiments, the dosage form of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is 100-400 mg/bottle, preferably 100-300 mg/bottle, 150-350 mg/bottle, 200-350 mg/bottle, 200-300 mg/bottle or 250-300 mg/bottle.
优选地，所述抗PVRIG/抗TIGIT抗体或其药物组合物的剂型规格为约100mg/瓶、150mg/瓶、180mg/瓶、200mg/瓶、250mg/瓶、300mg/瓶、350mg/瓶或400mg/瓶；优选地，所述抗PVRIG/抗TIGIT抗体的剂型或其药物组合物为单剂量剂型，每剂包含能够给予患 者所述有效施用量的抗体量。Preferably, the dosage form of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is about 100 mg/bottle, 150 mg/bottle, 180 mg/bottle, 200 mg/bottle, 250 mg/bottle, 300 mg/bottle, 350 mg/bottle or 400 mg/bottle; preferably, the dosage form of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is a single-dose dosage form, each dose comprising a single dose that can be administered to a patient. The amount of antibody that is effectively administered is preferably an amount of an antibody that is effectively administered.
在一些实施例中，所述抗PVRIG/抗TIGIT抗体或其药物组合物的给药频率为约每一周一次、每两周一次、每三周一次、每四周一次或一个月一次，优选为每周给药一次。In some embodiments, the anti-PVRIG/anti-TIGIT antibody or pharmaceutical composition thereof is administered approximately once a week, once every two weeks, once every three weeks, once every four weeks, or once a month, preferably once a week.
在一些实施例中，所述抗PVRIG/抗TIGIT抗体或其药物组合物通过静脉滴注、静脉注射、口服、皮下注射、肌内或瘤内注射给药，优选地，通过静脉输注给药。In some embodiments, the anti-PVRIG/anti-TIGIT antibody or pharmaceutical composition thereof is administered by intravenous drip, intravenous injection, oral administration, subcutaneous injection, intramuscular injection or intratumor injection, preferably, by intravenous infusion.
在一些实施例中，所述抗PVRIG/抗TIGIT抗体或其药物组合物的给药周期为一周、二周、三周、四周、两个月、三个月、四个月、五个月、半年或更长时间，任选地，每个给药周期的时间相同或不同，且每个给药周期之间的间隔相同或不同。In some embodiments, the administration cycle of the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is one week, two weeks, three weeks, four weeks, two months, three months, four months, five months, half a year or longer. Optionally, the time of each administration cycle is the same or different, and the interval between each administration cycle is the same or different.
在一些实施例中，所述患者根据RECIST 1.1版显示完全缓解或部分缓解。In some embodiments, the patient shows complete response or partial response according to RECIST version 1.1.
术语定义和说明Definitions and explanations of terms
除非本发明另外定义，与本发明相关的科学和技术术语应具有本领域普通技术人员所理解的含义。Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.
此外，除非本文另有说明，本文单数形式的术语应包括复数形式，复数形式的术语应包括单数形式。更具体地，如在本说明书和所附权利要求中所使用的，除非另外明确指出，否则单数形式“一种”和“这种”包括复数指示物。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.
本文术语“包括”、“包含”和“具有”之间可互换使用，旨在表示方案的包含性，意味着所述方案可存在除所列出的元素之外的其他元素。同时应当理解，在本文中使用“包括”、“包含”和“具有”描述，也提供“由……组成”方案。示例性地，“一种组合物，包括A和B”，应当理解为以下技术方案：由A和B组成的组合物，以及除A和B外，还含有其他组分的组合物，均落入前述“一种组合物”的范围内。The terms "include", "comprising", and "having" are used interchangeably herein, and are intended to indicate the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. It should also be understood that the use of "include", "comprising", and "having" in this article also provides a "consisting of" solution. Exemplarily, "a composition comprising A and B" should be understood as the following technical solution: a composition consisting of A and B, and a composition containing other components in addition to A and B, all fall within the scope of the aforementioned "a composition".
本文术语“和/或”在本文使用时，包括“和”、“或”和“由所属术语链接的要素的全部或任何其他组合”的含义。The term "and/or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".
本文术语“药物组合物”是指这样的制剂，其以允许包含在其中的活性成分的生物学活性有效的形式存在，并且不含有对施用所述药物组合物的受试者具有不可接受的毒性的另外的成分。药物组合物的目的是保持抗体活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本文中，“药物组合物”和“制剂”并不互相排斥。在一些实施方式中，本文术语“药物组合物”包括抗体，抗体衍生分子等。The term "pharmaceutical composition" herein refers to a preparation that exists in a form that allows the biological activity of the active ingredient contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to the subject to whom 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, etc.
本文术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of disease severity, 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 or complete), whether detectable or undetectable. Subjects in need of treatment include those who already have a condition or disease, as well as those who are susceptible to a condition or disease or those for whom a condition or disease is to be prevented. When referring to terms such as slow down, alleviate, weaken, alleviate, and relieve, their meanings also include elimination, disappearance, non-occurrence, and the like.
本文术语“患者”是指接受对如本发明所述的特定疾病或病症的治疗的人类。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。 The term "patient" herein refers to a human being receiving treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (eg, monkeys), or non-primate mammals receiving treatment for a disease or condition.
本文术语“肿瘤”指向或描述哺乳动物中典型地以不受调节的细胞生长为特征的生理状况。此定义中包括良性和恶性肿瘤。本文术语“肿瘤”或“瘤”是指所有赘生性(neoplastic)细胞生长和增殖,无论是恶性的还是良性的,及所有癌前(pre-cancerous)和癌性细胞和组织。术语“癌症”和“肿瘤”在本文中提到时并不互相排斥。The term "tumor" herein refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant tumors are included in this definition. The term "tumor" or "neoplasm" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.
本文所用术语“剂量”是引发治疗效果的药物的量。除非另有说明，否则剂量与游离形式的药物的量有关。如果药物是可药用盐形式，药物的量与游离形式的药物的量相比成比例地增加。例如，剂量将在产品包装或产品信息单中声明。As used herein, the term "dose" is the amount of a drug that elicits a therapeutic effect. Unless otherwise indicated, the dose is related to the amount of the drug in free form. If the drug is in the form of a pharmaceutically acceptable salt, the amount of the drug is increased proportionally compared to the amount of the drug in free form. For example, the dose will be stated on the product packaging or in a product information sheet.
术语“TIGIT”、“具有Ig和ITIM域的T细胞免疫受体”、“TIGIT抗原”、“Vstm3”和“WUCAM”可互换使用，并且包括各种哺乳动物同种型，例如人Tigit、人Tigit的直系同源物、和包含Tigit内的至少一个表位的类似物，以及具有至少一个与TIGIT共有的表位的类似物。TIGIT(例如人TIGIT)的氨基酸序列、以及编码其的核苷酸序列是本领域已知的。The terms "TIGIT," "T cell immunoreceptor having Ig and ITIM domains," "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 it are known in the art.
本文术语“PVRIG”或“PVRIG蛋白质”可以任选地包括任何这类蛋白质或其变异体、结合物或片段，包括(但不限于)如本文所述的已知或野生型PVRIG，以及任何天然产生的剪接变异体、氨基酸变异体或同工型，并且尤其是PVRIG的ECD片段。结合到PVRIG并且防止被PVRL2活化(例如，最常通过阻断PVRIG和PVLR2的相互作用)的“抗PVRIG抗体”(包括抗原结合片段)用于增强T细胞和/或NK细胞活化并且用于治疗疾病，如癌症和病原体感染。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 variant, amino acid variant or isoform, 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 of PVRIG and PVLR2) are used to enhance T cell and/or NK cell activation and to treat diseases such as cancer and pathogen infection.
本文术语“抗PVRIG/抗TIGIT抗体”和“双特异性PVRIG/TIGIT抗体”和“抗PVRIG/抗TIGIT双特异性抗体”可互换地使用，本发明的抗PVRIG/抗TIGIT双特异性抗体特异性地结合于人类TIGIT，并且优选是人类TIGIT的ECD，以及PVRIG，并且再优选是人类PVRIG的ECD。The terms "anti-PVRIG/anti-TIGIT antibody" and "bispecific PVRIG/TIGIT antibody" and "anti-PVRIG/anti-TIGIT bispecific antibody" are used interchangeably herein, and the anti-PVRIG/anti-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.
本文术语“特异性结合”是指抗原结合分子(例如抗体)通常以高亲和力特异性结合抗原和实质上相同的抗原，但不以高亲和力结合不相关抗原。亲和力通常以平衡解离常数(equilibrium dissociation constant，KD)来反映，其中较低KD表示较高亲和力。以抗体为例，高亲和力通常指具有约1×10-7M或更低、约1×10-8M或更低、约1×10-9M或更低、约1×10-10M或更低、1×10-11M或更低或1×10-12M或更低的KD。KD计算方式如下：KD＝Kd/Ka，其中Kd表示解离速率，Ka表示结合速率。可采用本领域周知的方法测量平衡解离常数KD，如表面等离子共振(例如Biacore)或平衡透析法测定。The term "specific binding" herein means that an antigen-binding molecule (e.g., an antibody) typically specifically binds to an antigen and substantially the same antigen with high affinity, but does not bind to unrelated antigens with high affinity. Affinity is typically reflected by an equilibrium dissociation constant (KD), wherein a lower KD indicates a higher affinity. Taking antibodies as an example, high affinity typically refers to a KD of about 1×10 -7 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, 1×10 -11 M or less, or 1×10 -12 M or less. KD is calculated as follows: KD=Kd/Ka, wherein Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured by methods well known in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis.
本文术语“抗原结合分子”按最广义使用，是指特异性结合抗原的分子。示例性地，抗原结合分子包括但不限于抗体或抗体模拟物。“抗体模拟物”是指能够与抗原特异性结合，但与抗体结构无关的有机化合物或结合域，示例性地，抗体模拟物包括但不限于affibody、affitin、affilin、经设计的锚蛋白重复蛋白(DARPin)、核酸适体或Kunitz型结构域肽。The term "antigen binding molecule" is used in the broadest sense herein 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" refers to organic compounds or binding domains that are able to specifically bind to an antigen but are unrelated to the antibody structure, and 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.
本文术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。 此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer et al.,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque et al.,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。The term "antibody" herein is used in the broadest sense and refers to a polypeptide or polypeptide combination that contains sufficient sequence from the variable region of the immunoglobulin heavy chain and/or sufficient sequence from the variable region of the immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various 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 contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, 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 transplant CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, etc.
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将本文“免疫球蛋白”分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。The "antibody" herein includes a typical "four-chain antibody", which belongs to an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to a polypeptide chain, which is composed of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from N-terminal to C-terminal; and, when the full-length antibody is an IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminal to C-terminal; the heavy chains and the heavy chains, and the heavy chains and the light chains are connected by disulfide bonds to form a "Y"-shaped structure. Due to the different amino acid compositions and arrangement orders of the constant regions of the heavy chains of immunoglobulins, their antigenicity is also different. Based on this, the "immunoglobulins" in this article can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of the disulfide bonds of the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into κ chains or λ chains by different constant regions. Each of the five types of Ig can have κ chains or λ chains.
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等骆驼科动物产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。The term "antibody" in this article also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by camelids such as dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanicoes (Lama guanicoe) and alpacas (Vicugna pacos), and immunoglobulin new antigen receptors (Ig new antigen receptors, IgNARs) found in cartilaginous fish such as sharks.
如本文所用，术语“重链抗体”是指缺乏常规抗体的轻链的抗体。该术语具体包括但不限于在不存在CH1结构域的情况下包含VH抗原结合结构域以及CH2和CH3恒定结构域的同型二聚体抗体。As used herein, the term "heavy chain antibody" refers to an antibody lacking the light chain of a conventional antibody. The term specifically includes, but is not limited to, a homodimeric antibody comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
如本文所用，术语“纳米抗体”是指骆驼体内存在天然的缺失轻链的重链抗体，克隆其可变区可以得到只有重链可变区组成的单域抗体，也称为VHH(Variable domain of heavy chain of heavy chain antibody)，它是最小的功能性抗原结合片段。As used in this article, the term "nanoantibody" refers to the natural heavy chain antibody lacking light chain that exists in camels. Cloning its variable region can obtain a single domain antibody consisting of only the heavy chain variable region, also called VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
本文术语“纳米抗体(nanobody)”、“单域抗体”(single domain antibody,sdAb)具有相同的含义并可互换使用，是指克隆重链抗体的可变区，构建仅由一个重链可变区组成的单域抗体，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的重链抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。In this article, the terms "nanobody" and "single domain antibody" (sdAb) have the same meaning and can be used interchangeably. They refer to cloning the variable region of a heavy chain antibody to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, a heavy chain antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.
关于“重链抗体”和“纳米抗体”的进一步描述可参见：Hamers-Casterman等，Nature.1993；363；446-8；Muyldermans的综述文章(Reviews inMolecular Biotechnology 74：277-302，2001)；以及以下专利申请，其被作为一般背景技术提及：WO 94/04678，WO 95/04079和WO 96/34103；WO94/25591，WO 99/37681，WO 00/40968，WO 00/43507，WO 00/65057，WO 01/40310，WO 01/44301，EP 1134231和WO 02/48193；WO97/49805，WO 01/21817，WO 03/035694，WO 03/054016和WO 03/055527；WO 03/050531；WO  01/90190；WO03/025020；以及WO 04/041867，WO 04/041862，WO 04/041865，WO 04/041863，WO 04/062551，WO 05/044858，WO 06/40153，WO 06/079372，WO 06/122786，WO 06/122787和WO 06/122825以及这些申请中提到的其他现有技术。For further description of "heavy chain antibodies" and "nanobodies", see: Hamers-Casterman et al., Nature. 1993; 363; 446-8; Muyldermans' review article (Reviews in Molecular Biotechnology 74: 277-302, 2001); and the following patent applications, which are mentioned as general background technology: WO 94/04678, WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527; WO 03/050531; WO 01/90190; WO03/025020; and WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825 and other prior art mentioned in these applications.
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。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, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).
本文“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。"Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.
本文术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转殖基因动物的方法和其它本领域已知的方法。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 generated during the production process of the preparation, such variants are usually 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 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 production of the antibody or antigen-binding molecule 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 using transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.
本文术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。The term "monospecific" as used herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.
本文术语“多特异性”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。The term "multispecific" herein refers to having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of different antigens. Thus, terms such as "bispecific", "trispecific", "tetraspecific" and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
本文术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。The term "valent" herein refers to the presence of a specified number of binding sites in an antibody/antigen binding molecule. Thus, the terms "monovalent", "divalent", "tetravalent" and "hexavalent" refer to the presence of one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively.
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。"Full length antibody," "intact antibody," and "intact antibody" are used interchangeably herein and refer to antibodies having a structure substantially similar to that of a native antibody.
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2、Fd、Fv、scFv、双抗体(diabody)和单域抗体。"Antigen binding fragment" and "antibody fragment" are used interchangeably herein, and they do not have the entire structure of a complete antibody, but only contain a partial or partial variant of a complete antibody, and the partial or partial variant 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, diabody and single domain antibody.
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab'片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫 醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。Papain digestion of intact antibodies generates two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is a fragment in which the cysteine residues of the constant domains carry a free sulfur Pepsin treatment generates a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and a portion of the Fc region.
本文术语“Fd”是指由VH和CH1结构域组成的抗体。本文术语“Fv”是指由单臂VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。The term "Fd" herein refers to an antibody consisting of a VH and CH1 domain. The term "Fv" herein refers to an antibody fragment consisting of a single-arm VL and VH domain. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to the antibody. However, even a single variable region (e.g., a Fd fragment, which contains only three CDRs specific for an antigen) can recognize and bind to an antigen, although its affinity may be lower than that of a complete binding site.
本文术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).
本文术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。The term "diabody" herein refers to an antibody whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
本文术语“裸抗体”是指不与治疗剂或示踪剂缀合的抗体；术语“缀合抗体”是指与治疗剂或示踪剂缀合的抗体，优选地，所述治疗剂选自药物、毒素、放射性同位素、化疗药或免疫调节剂，所述示踪剂选自放射学造影剂、顺磁离子、金属、荧光标记、化学发光标记、超声造影剂和光敏剂。The term "naked antibody" herein refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term "conjugated antibody" refers to an antibody conjugated to a therapeutic agent or tracer, preferably, the therapeutic agent is selected from a drug, a toxin, a radioisotope, a chemotherapeutic agent or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent and a photosensitizer.
本文术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt et al.,Kuby Immunology,6th ed.,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成： FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。The term "variable region" herein refers to the region of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen, and "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 natural antibodies (VH and VL, respectively) generally have similar structures, each domain comprising four conserved framework regions (FR) and three hypervariable regions (HVR). See, e.g., 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 usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This part is also called the complementarity determining region because it can form precise complementarity with the antigen epitope in terms of spatial structure. Among them, the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" or "FR region" are interchangeable and refer to those amino acid residues in the heavy chain variable region or light chain variable region of the antibody except for the CDR. Usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
对于CDR的进一步描述，参考Kabat等人,J.Biol.Chem.,252:6609-6616(1977)；Kabat等人,美国卫生与公共服务部，“Sequences of proteins of immunological interest”(1991)；Chothia等人,J.Mol.Biol.196:901-917(1987)；Al-Lazikani B.等人,J.Mol.Biol.,273:927-948(1997)；MacCallum等人,J.Mol.Biol.262:732-745(1996)；Abhinandan和Martin,Mol.Immunol.,45:3832-3839(2008)；Lefranc M.P.等人,Dev.Comp.Immunol.,27:55-77(2003)；以及Honegger和Plückthun,J.Mol.Biol.,309:657-670(2001)。本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。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., 273:9 27-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc M.P. et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDR" herein can be annotated and defined by methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system, and the tool websites used include but are 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). CDR herein includes overlaps and subsets of amino acid residues defined in different ways.
本文术语“Kabat编号系统”通常是指由ElvinA.Kabat提出的免疫球蛋白比对及编号系统(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。The term "Kabat numbering system" in this article generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：The term "conservative amino acid" herein generally refers to amino acids belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). Exemplarily, the amino acids within each of the following groups are conservative amino acid residues of each other, and the replacement of the amino acid residues within the group is a replacement of conservative amino acids:
示例性地，以下六组是被认为是互为保守性置换的氨基酸的实例：Illustratively, the following six groups are examples of amino acids that are considered to be conservative substitutions for each other:
1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)；1) Alanine (A), serine (S), threonine (T);
2)天冬氨酸(D)、谷氨酸(E)；2) Aspartic acid (D), Glutamic acid (E);
3)天冬酰胺(N)、谷氨酰胺(Q)；3) Asparagine (N), glutamine (Q);
4)精氨酸(R)、赖氨酸(K)、组氨酸(H)；4) Arginine (R), Lysine (K), Histidine (H);
5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V)；和5) isoleucine (I), leucine (L), methionine (M), valine (V); and
6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。6) Phenylalanine (F), tyrosine (Y), tryptophan (W).
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。The term "identity" herein can be calculated in the following manner: to determine the percentage of "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 comparison 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, the molecules are identical at this position.
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。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.
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百 分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the percent identity between two amino acid sequences can be determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at www.gcg.com) that has been integrated into the GAP program of the GCG software package, using 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. Score. As another example, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com) using the 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. A particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。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.
额外地或备选地，可以进一步使用本发明所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。例如，可以使用Altschul等人,(1990)J.Mol.Biol.215:403-10的NBLAST及XBLAST程序(版本2.0)执行此类检索。BLAST核苷酸检索可以用NBLAST程序，评分＝100、字长度＝12执行，以获得与本发明核酸分子同源的核苷酸序列。BLAST蛋白质检索可以用XBLAST程序、评分＝50、字长度＝3执行，以获得与本发明蛋白质分子同源的氨基酸序列。为了出于比较目的获得带空位的比对结果，可以如Altschul等人,(1997)Nucleic Acids Res.25:3389-3402中所述那样使用空位BLAST。当使用BLAST和空位BLAST程序时，可以使用相应程序(例如，XBLAST和NBLAST)的默认参数。参见www.ncbi.nlm.nih.gov。Additionally or alternatively, the nucleic acid sequences and protein sequences described in the present invention 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. 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.
本文术语“单剂量剂型”是指每剂包含单次给予受试者或患者有效施用量的药物。示例性地，单剂量剂型中所含的药物量可通过有效剂量换算。例如，按mg/m2计算的有效施用量，可根据人的体表面积，将单次的有效施用量换算成为单剂量剂型中每剂药的药物含量。例如，按mg/kg计算的有效施用量，可根据人的体重，将单次的有效施用量换算成单剂量剂型中每剂药的药物含量。另外也可以采用固定剂量给药，即不根据体重，一次性给予患者一定剂量的药物。固定剂量给药可以换算成为单剂量剂型中每剂药的药物含量，例如，固定给药剂量为150mg，其可以是单剂量剂型中每剂药的药物含量。同时，剂型可以是注射剂、片剂、粘合剂、混悬剂、气雾剂、颗粒剂、胶囊剂、膏剂或栓剂等。优选的，本文所述的抗TIGIT/抗PVRIG抗体可以以固定剂量的形式施用。The term "single-dose dosage form" herein refers to a drug that contains an effective amount of drug for a single administration to a subject or patient. Exemplarily, the amount of drug contained in a single-dose dosage form can be converted by an effective dose. For example, the effective amount calculated in mg/ m2 can be converted into the drug content of each dose of drug in a single-dose dosage form based on the body surface area of a person. For example, the effective amount calculated in mg/kg can be converted into the drug content of each dose of drug in a single-dose dosage form based on the body weight of a person. In addition, a fixed dose can also be used, that is, a certain dose of drug is given to the patient at one time regardless of body weight. Fixed dose administration can be converted into the drug content of each dose of drug in a single-dose dosage form, for example, a fixed dose of 150 mg, which can be the drug content of each dose of drug in a single-dose dosage form. At the same time, the dosage form can be an injection, tablet, adhesive, suspension, aerosol, granule, capsule, ointment or suppository, etc. Preferably, the anti-TIGIT/anti-PVRIG antibody described herein can be administered in the form of a fixed dose.
本文术语“G4S连接肽”是指甘氨酸(G)和丝氨酸(S)的GS组合，用于将多个蛋白连接在一起形成融合蛋白。常用的GS组合是(GGGGS)n，通过改变n的大小来改变接头序列的长度。同时，甘氨酸和丝氨酸还可以通过其他组合产生不同的接头序列，比如在本发明中使用的(G4S)4Linker，GS组合为GGGGS。The term "G4S linker peptide" herein refers to a GS combination of glycine (G) and serine (S), which is used to link multiple proteins together to form a fusion protein. A commonly used GS combination is (GGGGS)n, and the length of the linker sequence is changed by changing the size of n. At the same time, glycine and serine can also produce different linker sequences through other combinations, such as the (G4S)4Linker used in the present invention, the GS combination is GGGGS.
本文术语“不良反应”(AE)是与使用医学治疗相关的任何不利的和通常无意的或不期望的迹象、症状或疾病。例如，不良反应可能与在响应治疗时免疫系统的激活或免疫系统细胞的扩增相关。医学治疗可以具有一种或多种相关的AE，并且每种AE可以具有相同或不同的严重性水平。The term "adverse effect" (AE) herein is any unfavorable and generally unintended or undesirable sign, symptom or disease associated with the use of a medical treatment. For example, an adverse effect may be associated with activation of the immune system or expansion of immune system cells in response to a treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity.
本文术语“总生存期(Overall Survival，OS)”指从随机期至任何原因导致死亡的期。末次随访时仍存活的受试者，其OS以末次随访时间计为数据删失。失访的受试者，其OS以失访前末次证实存活时间计为数据删失。数据删失的OS定义为从随机分组到删失的时间。 The term "overall survival (OS)" in this article refers to the period from the randomization period to death from any cause. For subjects who are still alive at the last follow-up, their OS is calculated as data loss based on the last follow-up time. For subjects who are lost to follow-up, their OS is calculated as data loss based on the last confirmed survival time before loss of follow-up. OS with data loss is defined as the time from randomization to censoring.
本文术语“无进展生存(Progression Free Survival，PFS)”指从随机化至首次疾病进展(影像学)的时间，如果在疾病进展前因任何原因死亡的患者，则为随机化至死亡的时间。没有出现疾病进展或死亡的受试者，以最后一次影像学评价日期为删失日期。基线后没有进行影像学评价的受试者，以随机化日期删失。PFS的分析采用Kaplan-Meier估计中位无进展生存期(mPFS)及其95％CI，并绘制生存曲线图。The term "Progression Free Survival (PFS)" in this article refers to the time from randomization to the first disease progression (radiology). If the patient died of any cause before disease progression, it is the time from randomization to death. For subjects without disease progression or death, the date of the last imaging evaluation is used as the censoring date. Subjects who did not undergo imaging evaluation after baseline were censored at the randomization date. The Kaplan-Meier analysis of PFS estimated the median progression-free survival (mPFS) and its 95% CI, and a survival curve was drawn.
本文术语“客观缓解率(Objective response rate，ORR)”指肿瘤缩小达到一定并且保持一定时间的病人的比例，包含了CR和PR的病例。采用实体瘤缓解评估标准(RECIST 1.1标准)来评定肿瘤客观缓解。受试者在基线时必须伴有可测量的肿瘤病灶，疗效评定标准根据RECIST 1.1标准分为完全缓解(CR)、部分缓解(PR)、稳定(SD)、进展(PD)。The term "objective response rate (ORR)" in this article refers to the proportion of patients whose tumors shrink to a certain level and remain shrinking for a certain period of time, including cases of CR and PR. The solid tumor response evaluation criteria (RECIST 1.1 standard) are used to assess the objective response of tumors. Subjects must have measurable tumor lesions at baseline, and the efficacy evaluation criteria are divided into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD) according to the RECIST 1.1 standard.
本文术语“疾病控制率(Disease Control Rate，DCR)”指经确认的完全缓解、部分缓解和疾病稳定(≥8周)病例数在可评价疗效患者中的百分比。The term "Disease Control Rate (DCR)" in this article refers to the percentage of confirmed complete remission, partial remission, and stable disease (≥8 weeks) among patients who can be evaluated for efficacy.
本文术语“完全缓解(Complete response，CR)”：所有靶病灶消失，无新病灶出现。The term "complete response (CR)" in this article means that all target lesions disappear without the appearance of new lesions.
本文术语“部分缓解(Partial Remission，PR)”：靶病灶直径之和比基线水平减少至少30％。The term "partial remission (PR)" in this article is: the sum of the target lesion diameters is reduced by at least 30% compared with the baseline level.
本文术语“疾病进展(Progressive disease，PD)”：以整个实验研究过程中所有测量的靶病灶直径之和的最小值为参照，直径和相对增加至少20％(如果基线测量值最小就以基线值为参照)。The term "progressive disease (PD)" in this article is: the minimum value of the sum of all target lesion diameters measured during the entire experimental study is used as a reference, and the relative increase in the sum of diameters is at least 20% (if the baseline measurement value is the smallest, the baseline value is used as a reference).
本文术语“疾病稳定(Stable Disease，SD)”：靶病灶减小的程度没达到PR，增加的程度也没达到PD水平，介于两者之间，研究时可以直径之和的最小值作为参考。The term "stable disease (SD)" in this article means that the reduction of target lesions has not reached the level of PR, and the increase has not reached the level of PD, but is somewhere in between. The minimum value of the sum of diameters can be used as a reference for research.
本文术语“免疫治疗”是指通过包括诱导、增强、抑制或以其他方式修饰免疫反应的方法治疗患有疾病或具有感染或遭受疾病复发风险的受试者。受试者的“治疗”或“疗法”是指对受试者进行的任何类型的干预或过程，或给与受试者活性剂，目的在于逆转、缓解、改善、减缓或预防症状、并发症或病症的发作、进展、严重性或复发，或与疾病相关的生化指标。The term "immunotherapy" herein refers to treating a subject who has a disease or is at risk of infection or recurrence of a disease by methods including inducing, enhancing, suppressing or otherwise modifying an immune response. "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the purpose of reversing, alleviating, improving, slowing down or preventing the onset, progression, severity or recurrence of symptoms, complications or conditions, or biochemical indicators associated with the disease.
术语“给药方式”、“给药方案”可互换使用，是指本发明组合中每一治疗剂的使用剂量及时间。The terms "administration method" and "administration regimen" are used interchangeably to refer to the dosage and timing of each therapeutic agent in the combination of the present invention.
本文术语“RECIST 1.1疗效标准”是指Eisenhauver等人、E.A.等人，Eur.J Cancer 45:228-247(2009)基于所测量反应的背景针对靶标损伤或非靶标损伤所述的定义。在免疫治疗之前，其是实体肿瘤疗效评估最常用的标准。The term "RECIST 1.1 efficacy criteria" herein refers to the definition described by Eisenhauver et al., E.A. et al., Eur. J Cancer 45:228-247 (2009) for target lesions or non-target lesions based on the context of the measured response. Prior to immunotherapy, it was the most commonly used criterion for efficacy assessment in solid tumors.
本文术语“ECOG”评分标准，是从患者的体力来了解其一般健康状况和对治疗耐受能力的指标。ECOG体力状况评分标准记分：0分、1分、2分、3分、4分和5分。评分为0是指活动能力完全正常，与起病前活动能力无任何差异。评分为1是指能自由走动及从事轻体力活动，包括一般家务或办公室工作，但不能从事较重的体力活动。The term "ECOG" scoring standard in this article is an indicator of the patient's general health status and tolerance to treatment based on his or her physical strength. The ECOG physical status scoring standard is scored as follows: 0, 1, 2, 3, 4, and 5. A score of 0 means that the ability to move is completely normal, with no difference from the ability to move before the onset of the disease. A score of 1 means that the patient can move freely and engage in light physical activities, including general housework or office work, but cannot engage in heavier physical activities.
缩略语Abbreviations
贯穿于本发明的说明书及实施例中，使用以下缩略语：





Throughout the description and embodiments of the present invention, the following abbreviations are used:





附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型中各受试组肿瘤生长曲线Figure 1 Tumor growth curves of each test group in the humanized PBMC-B-NDG mouse Detroit 562 cell transplant tumor model
图2人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型中各受试组给药后小鼠体重变化情况Figure 2 Changes in body weight of mice in each test group after administration in the humanized PBMC-B-NDG mouse Detroit 562 cell transplant tumor model
图3人源化PBMC-B-NDG小鼠A375细胞移植瘤模型中各受试组肿瘤生长曲线。G1：PBS；G2：10mg/kg Atezolizumab；G3：2.57mg/kg TIGIT-002-H4L3；G4：1.36mg/kg PVRIG-50H1b；G5：8.57mg/kg TIGIT-002-H4L3；G6：4.53mg/kg PVRIG-50H1b；G7：3mg/kg LC-BsAb-002；G8：10mg/kg LC-BsAb-002Figure 3 Tumor growth curves of each test group in the humanized PBMC-B-NDG mouse A375 cell transplant tumor model. G1: PBS; G2: 10mg/kg Atezolizumab; G3: 2.57mg/kg TIGIT-002-H4L3; G4: 1.36mg/kg PVRIG-50H1b; G5: 8.57mg/kg TIGIT-002-H4L3; G6: 4.53mg/kg PVRIG-50H1b; G7: 3mg/kg LC-BsAb-002; G8: 10mg/kg LC-BsAb-002
图4人源化PBMC-B-NDG小鼠A375细胞移植瘤模型中各受试组给药后小鼠体重变化情况。G1：PBS；G2：10mg/kg Atezolizumab；G3：2.57mg/kg TIGIT-002-H4L3；G4：1.36mg/kg PVRIG-50H1b；G5：8.57mg/kg TIGIT-002-H4L3；G6：4.53mg/kg PVRIG-50H1b；G7：3mg/kg LC-BsAb-002；G8：10mg/kg LC-BsAb-002Figure 4 Changes in body weight of mice in each test group after administration in the humanized PBMC-B-NDG mouse A375 cell transplant tumor model. G1: PBS; G2: 10mg/kg Atezolizumab; G3: 2.57mg/kg TIGIT-002-H4L3; G4: 1.36mg/kg PVRIG-50H1b; G5: 8.57mg/kg TIGIT-002-H4L3; G6: 4.53mg/kg PVRIG-50H1b; G7: 3mg/kg LC-BsAb-002; G8: 10mg/kg LC-BsAb-002
图5使用BOIN设计的试验实施流程图Figure 5 Experimental implementation flow chart using BOIN design
具体实施方式Detailed ways
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
本发明实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。The embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.
实施例1抗TIGIT/抗PVRIG抗体的构建、表达纯化、制剂制备Example 1 Construction, expression, purification and preparation of anti-TIGIT/anti-PVRIG antibodies
1.1抗TIGIT/抗PVRIG抗体的构建1.1 Construction of anti-TIGIT/anti-PVRIG antibodies
用G4S连接肽将抗PVRIG人源化VHH抗体PVRIG-A50-H1b连接到抗TIGIT人源化单克隆抗体TIGIT-002-H4L3重链的N端，产生抗PVRIG×TIGIT人源化双特异性抗体，命名为LC-BsAb-002。表1所示为LC-BsAb-002重链融合多肽(HC)和轻链多肽(LC)的序列，表2所示为LC-BsAb-002的可变区序列，表3所示为LC-BsAb-002按照Kabat划分的CDR序列。The anti-PVRIG humanized VHH antibody PVRIG-A50-H1b was connected to the N-terminus of the heavy chain of the anti-TIGIT humanized monoclonal antibody TIGIT-002-H4L3 using a G4S connecting peptide to generate an anti-PVRIG×TIGIT humanized bispecific antibody named LC-BsAb-002. Table 1 shows the sequences of the heavy chain fusion polypeptide (HC) and light chain polypeptide (LC) of LC-BsAb-002, Table 2 shows the variable region sequence of LC-BsAb-002, and Table 3 shows the CDR sequence of LC-BsAb-002 divided according to Kabat.
表1抗TIGIT/抗PVRIG抗体LC-BsAb-002融合多肽序列
Table 1 Anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 fusion polypeptide sequence
表2抗TIGIT/抗PVRIG抗体的可变区序列
Table 2 Variable region sequences of anti-TIGIT/anti-PVRIG antibodies
表3抗TIGIT/抗PVRIG抗体的KABAT分析结果
Table 3 KABAT analysis results of anti-TIGIT/anti-PVRIG antibodies
1.2抗TIGIT/抗PVRIG抗体的表达1.2 Expression of anti-TIGIT/anti-PVRIG antibodies
采用基因工程技术将抗TIGIT/抗PVRIG抗体LC-BsAb-002基因转染至CHO-K1细胞中，并经过MSX压力筛选出产量较高的克隆，在CD CHO培养基中进行传代培养。抗TIGIT/抗PVRIG抗体运用分批补料培养方式进行表达。基础培养基为奥普迈的CHO CDP9，培养周期14-16天，反应器控制参数为pH 6.6～7.2，溶解氧(DO)为≥20％，转速75RPM～80RPM，初始培养温度36.0℃～37.0℃；当培养至第6天时，降低培养温度至33.5℃～34.5℃直到收获。The anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 gene was transfected into CHO-K1 cells using genetic engineering technology, and clones with higher yields were selected through MSX pressure screening and subcultured in CD CHO medium. Anti-TIGIT/anti-PVRIG antibodies were expressed using a fed-batch culture method. The basal culture medium was Opma's CHO CDP9, the culture cycle was 14-16 days, the reactor control parameters were pH 6.6-7.2, dissolved oxygen (DO) was ≥20%, the speed was 75RPM-80RPM, and the initial culture temperature was 36.0℃-37.0℃; when the culture reached the 6th day, the culture temperature was lowered to 33.5℃-34.5℃ until harvest.
补料培养基为奥普迈的XF04和CD FS08，从Day3开始隔天补，补料体积分别是体积的5％和0.5％，直至收获。The feed media were XF04 and CD FS08 from Opma, which were fed every other day starting from Day 3, with feed volumes of 5% and 0.5% of the volume respectively, until harvest.
1.3抗TIGIT/抗PVRIG抗体的纯化1.3 Purification of anti-TIGIT/anti-PVRIG antibodies
抗TIGIT/抗PVRIG抗体LC-BsAb-002的纯化采用多步骤层析、浓缩和过滤单元操作依次对抗体进行纯化。上清收获液经过Protein A亲和层析(AT ProteinA Diamond Plus)进行捕获。捕获后的抗体溶液采用低pH孵育处理以灭活潜在的病毒。抗体溶液中和后通过深层过滤除去沉淀。然后依次进行阴离子交换层析(Diamond Q)以去除HCD,HCP,脱落ProteinA等杂质，以及阳离子交换(Diamond S)层析以去除HCP和聚集体等杂质。通过纳滤膜包过滤以去除潜在的内外源病毒。之后用超滤膜包对抗体溶液进行浓缩，缓冲液置换，从而完成纯化，获得抗TIGIT/抗PVRIG抗体蛋白原液。Purification of anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 uses multi-step chromatography, concentration and filtration unit operations to purify the antibody in sequence. The supernatant harvest is captured by Protein A affinity chromatography (AT Protein A Diamond Plus). The captured antibody solution is treated with low pH incubation to inactivate potential viruses. After neutralization of the antibody solution, the precipitate is removed by deep filtration. Then, anion exchange chromatography (Diamond Q) is 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 package to remove potential exogenous and exogenous viruses. The antibody solution is then concentrated and buffer exchanged using an ultrafiltration membrane package to complete the purification and obtain the anti-TIGIT/anti-PVRIG antibody protein stock solution.
1.4抗TIGIT/抗PVRIG抗体制剂的制备1.4 Preparation of anti-TIGIT/anti-PVRIG antibody preparations
筛选抗TIGIT/抗PVRIG抗体的制剂处方：设计不同的缓冲液体系、不同的辅料和不同的表面活性剂，经稳定性研究，筛选出最优处方。Screening of formulations for anti-TIGIT/anti-PVRIG antibodies: designing different buffer systems, different excipients and different surfactants, and screening out the optimal formulation through stability studies.
选择20mM醋酸-醋酸钠，pH5.0；8％(w/v)蔗糖；0.04％(w/v)PS80用于处方确认稳定性研究。包材选择10mL西林瓶，10mm胶塞和10mm铝塑盖。规格：50mg/mL抗PVRIG/抗TIGIT抗体LC-BsAb-002；装量：6mL/300mg。考察指标包括：外观、pH、蛋白浓度、动态光散射(DLS)、阳离子交换色谱(CEX)、纯度(分子排阻色谱法(SE-HPLC)、非还原十二烷基硫酸钠毛细管电泳(CE-SDS(NR&R))和结合活性(ELISA-Binding)。考察方案详见表4。20mM acetic acid-sodium acetate, pH 5.0; 8% (w/v) sucrose; 0.04% (w/v) PS80 were selected for the formulation confirmation stability study. The packaging material was 10mL vials, 10mm rubber stoppers and 10mm aluminum-plastic caps. Specification: 50mg/mL anti-PVRIG/anti-TIGIT antibody LC-BsAb-002; Filling volume: 6mL/300mg. The inspection indicators include: appearance, pH, protein concentration, dynamic light scattering (DLS), cation exchange chromatography (CEX), purity (molecular exclusion chromatography (SE-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS (NR&R)) and binding activity (ELISA-Binding). The inspection plan is detailed in Table 4.
表4处方确认稳定性考察方案
Table 4 Stability test plan for prescription confirmation
注：X＝外观，pH，蛋白浓度，DLS，iCIEF，SE-HPLC，CE-SDS(NR)，ELISA-Binding，不溶性微粒(MFI)。Note: X = appearance, pH, protein concentration, DLS, iCIEF, SE-HPLC, CE-SDS (NR), ELISA-Binding, insoluble particles (MFI).
结果显示，该处方的外观、pH、蛋白浓度、不溶性微粒、DLS粒径大小、结合活性在整个考察期间无明显变化，当样品在低温、室温和高温下储存时，CEX、SE-HPLC、CE-SDS考察显示该处方稳定性良好。The results showed that the appearance, pH, protein concentration, insoluble particles, DLS particle size and binding activity of the formulation did not change significantly during the entire investigation period. When the samples were stored at low temperature, room temperature and high temperature, CEX, SE-HPLC and CE-SDS investigations showed that the formulation had good stability.
最终处方确定为50mg/mL PVRIG/TIGIT双特异性抗体；20mM醋酸-醋酸钠，pH5.0；8％(w/v)蔗糖；0.04％(w/v)PS80。The final prescription was determined to be 50 mg/mL PVRIG/TIGIT bispecific antibody; 20 mM acetic acid-sodium acetate, pH 5.0; 8% (w/v) sucrose; 0.04% (w/v) PS80.
实施例2抗TIGIT/抗PVRIG抗体的功效：人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型中的治疗Example 2 Efficacy of anti-TIGIT/anti-PVRIG antibodies: Treatment in a humanized PBMC-B-NDG mouse Detroit 562 cell xenograft tumor model
将0.2mL含有5×106人PBMC的1640培养基静脉接种于每只小鼠。PBMC接种后3天，在每只小鼠的右侧腋下皮下接种0.1mL含5×106个Detroit 562人咽癌细胞的PBS。当肿瘤平均体积达到约84.38mm3时，选择36只肿瘤体积中等且PBMC人源化水平中等的小鼠入组。将小鼠分配至6个实验组：PBS组、Atezolizumab 8.27mg/kg组、LC-BsAb-002 1、3、10和30mg/kg组；在分组当天开始通过腹腔注射给药，当天记录为第0天，每周给药2次，即第0、3、7、10、14、17天各给药一次。0.2 mL of 1640 medium containing 5×10 6 human PBMCs was inoculated intravenously into each mouse. Three days after PBMC inoculation, 0.1 mL of PBS containing 5×10 6 Detroit 562 human pharyngeal carcinoma cells was inoculated subcutaneously in the right axilla of each mouse. When the average tumor volume reached approximately 84.38 mm 3 , 36 mice with medium tumor volume and medium PBMC humanization level were selected for enrollment. The mice were assigned to 6 experimental groups: PBS group, Atezolizumab 8.27 mg/kg group, LC-BsAb-002 1, 3, 10 and 30 mg/kg groups; administration began on the day of grouping by intraperitoneal injection, which was recorded as day 0, and was administered twice a week, i.e., once on days 0, 3, 7, 10, 14, and 17.
图1和表5显示，分组后第17天，PBS组(G1)的平均肿瘤体积为920.53mm3。Atezolizumab 8.27mg/kg组(G2)、LC-BsAb-002 30mg/kg组(G3)、10mg/kg组(G4)、3mg/kg组(G5)、1mg/kg组(G6)的平均肿瘤均显著低于PBS组。Atezolizumab 8.27mg/kg组的TGI为24.63％；LC-BsAb-00230、10、3和1mg/kg组的TGI分别为68.10％、60.07％、63.21％和62.32％，均显著优于Atezolizumab 8.27mg/kg(P<0.05)。这些结果表明，LC-BsAb-002在人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型中表现出显著的抗肿瘤药效，然而并未观察到剂量依赖性。Figure 1 and Table 5 show that on the 17th day after grouping, the average tumor volume of the PBS group (G1) was 920.53 mm 3 . The average tumors of the Atezolizumab 8.27 mg/kg group (G2), LC-BsAb-002 30 mg/kg group (G3), 10 mg/kg group (G4), 3 mg/kg group (G5), and 1 mg/kg group (G6) were significantly lower than those of the PBS group. The TGI of the Atezolizumab 8.27 mg/kg group was 24.63%; the TGI of the LC-BsAb-002 30, 10, 3, and 1 mg/kg groups were 68.10%, 60.07%, 63.21%, and 62.32%, respectively, which were significantly better than Atezolizumab 8.27 mg/kg (P<0.05). These results indicate that LC-BsAb-002 exhibited significant antitumor efficacy in the humanized PBMC-B-NDG mouse Detroit 562 cell xenograft tumor model, however, no dose-dependency was observed.
表5 LC-BsAb-002在人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型中的疗效
Table 5 Efficacy of LC-BsAb-002 in the Detroit 562 cell transplant tumor model of humanized PBMC-B-NDG mice
注：Note:
a.平均值±SEM。a. Mean ± SEM.
b.相对肿瘤生长率T/C％＝TRTV/CRTV×100％(TRTV：给药组平均RTV；CRTV：对照组平均RTV)。相对肿瘤体积，RTV＝Vt/V0(V0：第0天的肿瘤体积，Vt：第17天的肿瘤体积)。b. Relative tumor growth rate T/C% = TRTV/CRTV×100% (TRTV: average RTV of the drug administration group; CRTV: average RTV of the control group). Relative tumor volume, RTV = Vt/V0 (V0: tumor volume on day 0, Vt: tumor volume on day 17).
c.TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％。c.TGI(%)=[1-(Ti-T0)/(Vi-V0)]×100%.
d.通过双因素ANOVA分析数据,与G1 PBS相比，认为P<0.05具有统计学显著性，***：P<0.001.d. Data were analyzed by two-way ANOVA. P<0.05 was considered statistically significant compared with G1 PBS. *** : P<0.001.
图2和表6显示，与第0天相比，PBS组、Atezolizumab 8.27mg/kg组、LC-BsAb-002 30、10、3和1mg/kg组的平均体重变化分别为4.0％、-3.6％、-9.8％、-1.3％、-3.8％和-8.3％。尽管在LC-BsAb-002给药组中观察到体重减轻，但小鼠的状态和行为均正常，表明小鼠可耐受1mg/kg至30mg/kg剂量的LC-BsAb-002。Figure 2 and Table 6 show that the average body weight changes in the PBS group, Atezolizumab 8.27 mg/kg group, LC-BsAb-002 30, 10, 3, and 1 mg/kg groups were 4.0%, -3.6%, -9.8%, -1.3%, -3.8%, and -8.3%, respectively, compared with day 0. Although weight loss was observed in the LC-BsAb-002-administered groups, the state and behavior of the mice were normal, indicating that the mice could tolerate LC-BsAb-002 at doses of 1 mg/kg to 30 mg/kg.
表6 LC-BsAb-002对人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型小鼠体重和存活率的影响
Table 6 Effects of LC-BsAb-002 on body weight and survival rate of Detroit 562 cell transplanted tumor model mice in humanized PBMC-B-NDG mice
注：平均值±SEM。Note: Mean ± SEM.
综上，在本研究中，LC-BsAb-002在人源化PBMC-B-NDG小鼠Detroit 562细胞移植瘤模型中表现出显著抗肿瘤药效，30、10、3和1mg/kg组的TGI分别为68.10％、60.07％、63.21％和62.32％，均显著优于8.27mg/kg的Atezolizumab。在实验中，无动物停药。尽管在LC-BsAb-002给药组中观察到小鼠体重减轻，但小鼠的状态和行为正常，表明小鼠可耐受1mg/kg至30mg/kg的LC-BsAb-002。In summary, in this study, LC-BsAb-002 showed significant antitumor efficacy in the humanized PBMC-B-NDG mouse Detroit 562 cell transplant tumor model, and the TGI of the 30, 10, 3 and 1 mg/kg groups were 68.10%, 60.07%, 63.21% and 62.32%, respectively, which were significantly better than 8.27 mg/kg of Atezolizumab. In the experiment, no animals stopped taking the drug. Although weight loss was observed in the LC-BsAb-002-treated group, the mice were in normal condition and behavior, indicating that mice can tolerate 1 mg/kg to 30 mg/kg of LC-BsAb-002.
实施例3抗TIGIT/抗PVRIG抗体的功效：在人源化PBMC-B-NDG小鼠A375细胞移植瘤模型中的治疗Example 3 Efficacy of anti-TIGIT/anti-PVRIG antibodies: Treatment in a humanized PBMC-B-NDG mouse A375 cell xenograft model
在本实施例中，在每只小鼠的右侧腋下皮下接种0.1mL含5×106个A375人黑色素瘤细胞的PBS，以形成肿瘤。A375细胞接种后第二天，每只小鼠静脉接种0.2mL含5×106个人PBMC的1640培养基。当肿瘤平均体积达到约119.07mm3时，选择64只具有中等肿瘤体积和中等PBMC人源化水平的大于0.8％的小鼠入组。将小鼠分配至8个实验组：PBS组、Atezolizumab 10mg/kg组、TIGIT-002-H4L3 2.57mg/kg组、PVRIG-50H1b 1.36mg/kg组、TIGIT-002-H4L3 8.57mg/kg组、PVRIG-50H1b 4.53mg/kg组、LC-BsAb-002 3 mg/kg组和10mg/kg组；分组当天开始给药，当天记录为第0天，每周给药2次，即第0、4、8、11、15天各给药一次。In this example, 0.1 mL of PBS containing 5×10 6 A375 human melanoma cells was subcutaneously inoculated in the right axilla of each mouse to form a tumor. On the second day after A375 cell inoculation, each mouse was intravenously inoculated with 0.2 mL of 1640 culture medium containing 5×10 6 human PBMCs. When the average tumor volume reached about 119.07 mm 3 , 64 mice with medium tumor volume and medium PBMC humanization level greater than 0.8% were selected for enrollment. The mice were assigned to 8 experimental groups: PBS group, Atezolizumab 10 mg/kg group, TIGIT-002-H4L3 2.57 mg/kg group, PVRIG-50H1b 1.36 mg/kg group, TIGIT-002-H4L3 8.57 mg/kg group, PVRIG-50H1b 4.53 mg/kg group, LC-BsAb-002 3 mg/kg group and 10mg/kg group; drug administration began on the day of grouping, which was recorded as day 0, and was administered twice a week, i.e. once on days 0, 4, 8, 11, and 15.
分组后第15天，PBS组(G1)的平均肿瘤体积为1153.11mm3。与PBS组相比，10mg/kg LC-BsAb-002(G8)组肿瘤体积显著降低，TGI为47.02％(P＝0.0027)，而8.27mg/kg Atezolizumab(G2)、2.57和8.57mg/kg TIGIT-002-H4L3(G3和G5)、1.36和4.53mg/kg PVRIG-50H1b(G4和G6)及3mg/kg LC-BsAb-002(G7)均未能显著降低肿瘤体积(图3、表7)。On the 15th day after grouping, the average tumor volume of the PBS group (G1) was 1153.11 mm 3 . Compared with the PBS group, the tumor volume of the 10 mg/kg LC-BsAb-002 (G8) group was significantly reduced, with a TGI of 47.02% (P=0.0027), while 8.27 mg/kg Atezolizumab (G2), 2.57 and 8.57 mg/kg TIGIT-002-H4L3 (G3 and G5), 1.36 and 4.53 mg/kg PVRIG-50H1b (G4 and G6) and 3 mg/kg LC-BsAb-002 (G7) failed to significantly reduce the tumor volume ( Figure 3 , Table 7 ).
表7 LC-BsAb-002在人源化PBMC-B-NDG小鼠A375细胞移植瘤模型小鼠中的疗效
Table 7 Efficacy of LC-BsAb-002 in humanized PBMC-B-NDG mouse A375 cell transplant tumor model mice
a.平均值±SEM。a. Mean ± SEM.
b.相对肿瘤生长率T/C％＝TRTV/CRTV×100％(TRTV：给药组平均RTV；CRTV：对照组平均RTV)。相对肿瘤体积，RTV＝Vt/V0(V0：第0天的肿瘤体积，Vt：第17天的肿瘤体积)。b. Relative tumor growth rate T/C% = TRTV/CRTV×100% (TRTV: average RTV of the drug administration group; CRTV: average RTV of the control group). Relative tumor volume, RTV = Vt/V0 (V0: tumor volume on day 0, Vt: tumor volume on day 17).
c.TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％。c.TGI(%)=[1-(Ti-T0)/(Vi-V0)]×100%.
d.通过双因素ANOVA分析数据，与G1 PBS相比，P<0.05被视为具有统计学显著性，**：P<0.01。d. Data were analyzed by two-way ANOVA, P<0.05 was considered statistically significant compared with G1 PBS, ** : P<0.01.
与D0相比，PBS组、Atezolizumab 8.27mg/kg组、TIGIT-002-H4L3 2.57mg/kg组、PVRIG-50H1b 1.36mg/kg组、TIGIT-002-H4L3 8.57mg/kg组、PVRIG-50H1b 4.53mg/kg组、LC-BsAb-002 3和10mg/kg组的平均体重变化分别为4.8％、-2.8％、5.0％、5.6％、-3.4％、1.8％、-3.2％和1.7％(图4、表8)。在本研究中，无动物停药。尽管在LC-BsAb-002给药组中观察到体重减轻，但小鼠的状态和行为正常，表明小鼠可耐受3和10mg/kg剂量的LC-BsAb-002。Compared with D0, the mean body weight changes in the PBS group, Atezolizumab 8.27 mg/kg group, TIGIT-002-H4L3 2.57 mg/kg group, PVRIG-50H1b 1.36 mg/kg group, TIGIT-002-H4L3 8.57 mg/kg group, PVRIG-50H1b 4.53 mg/kg group, LC-BsAb-002 3 and 10 mg/kg groups were 4.8%, -2.8%, 5.0%, 5.6%, -3.4%, 1.8%, -3.2%, and 1.7%, respectively (Figure 4, Table 8). In this study, no animals were discontinued. Although weight loss was observed in the LC-BsAb-002-administered groups, the status and behavior of the mice were normal, indicating that the mice could tolerate LC-BsAb-002 at doses of 3 and 10 mg/kg.
表8 LC-BsAb-002人源化PBMC-B-NDG小鼠A375细胞移植瘤模型小鼠体重和存活率的影响

Table 8 Effect of LC-BsAb-002 on body weight and survival rate of mice in humanized PBMC-B-NDG mouse A375 cell transplant tumor model

注：平均值±SEM。Note: Mean ± SEM.
综上，10mg/kg LC-BsAb-002在人源化PBMC-B-NDG小鼠A375细胞移植瘤模型中表现出显著的抗肿瘤疗效，TGI为47.02％，优于等摩尔的anti-TIGIT、anti-PVRIG单药和Atezolizumab。在实验中，无动物停药，3和10mg/kg剂量下的LC-BsAb-002在小鼠中耐受良好。In summary, 10mg/kg LC-BsAb-002 showed significant anti-tumor efficacy in the humanized PBMC-B-NDG mouse A375 cell transplant tumor model, with a TGI of 47.02%, which was superior to equimolar anti-TIGIT, anti-PVRIG monotherapy and Atezolizumab. In the experiment, no animals were discontinued, and LC-BsAb-002 at doses of 3 and 10mg/kg was well tolerated in mice.
实施例4抗TIGIT/抗PVRIG抗体在晚期实体瘤受试者中的安全性、耐受性、药代动力学和初步抗肿瘤活性的首次人体、开放性、剂量递增I期研究Example 4 A first-in-human, open-label, dose-escalation phase I study of safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of anti-TIGIT/anti-PVRIG antibodies in subjects with advanced solid tumors
4.1研究目的4.1 Research objectives
本研究旨在评价抗TIGIT/抗PVRIG抗体单药治疗晚期实体瘤成人受试者的安全性和耐受性、药代动力学/药效学特征并评估初步疗效。This study aims to evaluate the safety and tolerability, pharmacokinetic/pharmacodynamic characteristics and preliminary efficacy of anti-TIGIT/anti-PVRIG antibody monotherapy in adult subjects with advanced solid tumors.
4.2研究设计4.2 Study design
4.2.1总体设计4.2.1 Overall design
第1部分(剂量递增和剂量递增扩展)：Part 1 (dose escalation and dose expansion):
该部分旨在评价抗TIGIT/抗PVRIG抗体的安全性、耐受性、药代动力学/药效学、免疫原性特征、初步抗肿瘤活性，并确立抗TIGIT/抗PVRIG抗体的RD。第1部分包括剂量递增和剂量递增扩展部分。This part aims to evaluate the safety, tolerability, pharmacokinetic/pharmacodynamics, immunogenicity characteristics, preliminary anti-tumor activity of anti-TIGIT/anti-PVRIG antibodies, and establish the RD of anti-TIGIT/anti-PVRIG antibodies. Part 1 includes dose escalation and dose escalation expansion parts.
第1部分(剂量递增)：计划入组标准治疗(SOC)失败或不适合标准治疗的晚期实体瘤受试者。将采用贝叶斯最优区间(BOIN)设计。合格受试者将从起始剂量10mg每周一次(QW)开始，在每个周期(28天)的D1、D8、D15、D22接受抗TIGIT/抗PVRIG抗体静脉输注(IV)。剂量递增计划为10、30、100、300和600mg，采用半对数递增策略，但最后2个剂量组除外，采用100％递增。Part 1 (Dose Escalation): It is planned to enroll subjects with advanced solid tumors who have failed or are not suitable for standard treatment (SOC). A Bayesian Optimal Interval (BOIN) design will be used. Eligible subjects will start with a starting dose of 10 mg once a week (QW) and receive anti-TIGIT/anti-PVRIG antibody intravenous infusion (IV) on D1, D8, D15, and D22 of each cycle (28 days). The dose escalation plan is 10, 30, 100, 300, and 600 mg, using a semi-logarithmic escalation strategy, except for the last 2 dose groups, which use a 100% increase.
MTD的目标DLT发生率设定为0.3，如因受试者过早退出导致某剂量水平PK特征考察不充分，可经研究者和申办方共同决议是否对该剂量水平补充入组受试者，实际入组患者数量将取决于达到MTD或最大给药剂量(MAD)的剂量水平以及已完成队列额外入组受试者的情况。The target DLT incidence rate of MTD is set at 0.3. If the PK characteristics of a certain dose level are not fully investigated due to premature withdrawal of subjects, the investigator and the sponsor may jointly decide whether to enroll additional subjects at this dose level. The actual number of patients enrolled will depend on the dose level that reaches the MTD or maximum administered dose (MAD) and the additional enrollment of subjects in the completed cohort.
在每个剂量水平的末例受试者完成1周期治疗后，安全性审查委员会(SRC)将根据总体数据(包括但不限于安全性、疗效、PK和PD)决定是否将剂量递增至下一剂量水平。第1部分剂量递增期间，根据安全性、耐受性和药代动力学，可允许探索基于申办方和主要研究者(PI)通过SRC共同决定的中间剂量水平或剂量超过600mg QW。可根据抗TIGIT/抗PVRIG抗体的累积数据考虑其他方案。 After the last subject at each dose level completes 1 cycle of treatment, the Safety Review Committee (SRC) will decide whether to escalate the dose to the next dose level based on the overall data (including but not limited to safety, efficacy, PK and PD). During the dose escalation period in Part 1, based on safety, tolerability and pharmacokinetics, it may be allowed to explore intermediate dose levels or doses exceeding 600 mg QW jointly decided by the sponsor and the principal investigator (PI) through the SRC. Other regimens may be considered based on the cumulative data of anti-TIGIT/anti-PVRIG antibodies.
第1部分(剂量递增扩展)：将根据剂量递增部分的结果决定剂量递增扩展部分。计划入组SOC失败或不适合标准治疗的晚期实体瘤受试者。可入组额外的受试者至已证明不超过MTD或MAD且SRC已宣布安全的回填剂量队列，以收集该剂量水平的额外安全性和PK数据。Part 1 (Dose Escalation Expansion): The dose escalation expansion part will be decided based on the results of the dose escalation part. It is planned to enroll subjects with advanced solid tumors who have failed SOC or are not suitable for standard treatment. Additional subjects may be enrolled in the backfill dose cohort that has been demonstrated to not exceed the MTD or MAD and has been declared safe by the SRC to collect additional safety and PK data at this dose level.
受试者在完成1周期治疗后可继续接受抗TIGIT/抗PVRIG抗体治疗，直至符合治疗终止标准。After completing one cycle of treatment, subjects can continue to receive anti-TIGIT/anti-PVRIG antibody treatment until they meet the treatment discontinuation criteria.
第1部分除了上述提到的受试者外，可能入组额外的受试者，以确定安全性和PK(例如，如遇特殊要求，可入组特定的剂量队列)。In addition to the subjects mentioned above, additional subjects may be enrolled in Part 1 to determine safety and PK (e.g., specific dose cohorts may be enrolled if special requirements are met).
第2部分(队列扩展)：Part 2 (Queue Expansion):
第2部分将在第1部分确定抗TIGIT/抗PVRIG抗体单药治疗的RD方案后开始，目的是更好地描述抗TIGIT/抗PVRIG抗体的安全性和PK，并评价研究药物的初步抗肿瘤作用。队列扩展人群的选择将基于最新数据，例如第1部分的结果和具有相同/相似作用机制的其他药物的累积新数据，包括但不限于以下癌症：非小细胞肺癌(NSCLC)、肝细胞癌(HCC)和其他实体瘤组。此外，如果在一个队列的亚组人群中证实了临床获益，SRC可能会限制亚组的入组。Part 2 will begin after the RD regimen of anti-TIGIT/anti-PVRIG antibody monotherapy is determined in Part 1, with the goal of better describing the safety and PK of anti-TIGIT/anti-PVRIG antibodies and evaluating the preliminary anti-tumor effects of the study drug. The selection of the cohort expansion population will be based on the latest data, such as the results of Part 1 and the accumulated new data of other drugs with the same/similar mechanism of action, including but not limited to the following cancers: non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC) and other solid tumor groups. In addition, if clinical benefit is confirmed in a subgroup population of a cohort, the SRC may limit the enrollment of the subgroup.
合格受试者将接受RD水平的抗TIGIT/抗PVRIG抗体静脉给药，直至符合治疗终止标准。Eligible subjects will receive RD levels of anti-TIGIT/anti-PVRIG antibodies intravenously until treatment discontinuation criteria are met.
4.2.2剂量递增规则4.2.2 Dose escalation rules
将采用贝叶斯最优区间(BOIN)设计确定MTD(如存在)和RD。仅第1周期内发生的DLT用于剂量探索。如图5所示，BOIN设计使用优化的以下规则，以最大限度地减少剂量分配错误的概率，指导剂量递增/递减：A Bayesian optimal interval (BOIN) design will be used to determine the MTD (if any) and RD. Only DLTs occurring within cycle 1 will be used for dose finding. As shown in Figure 5, the BOIN design uses the following rules optimized to minimize the probability of dose allocation errors to guide dose escalation/decrease:
●如果在当前剂量下观察到的DLT发生率≤0.236，则将剂量递增至下一个更高的剂量水平；If the observed DLT rate at the current dose is ≤ 0.236, escalate the dose to the next higher dose level;
●如果在当前剂量下观察到的DLT发生率>0.359，将剂量递减至下一个较低剂量水平；●If the observed DLT rate at the current dose is >0.359, reduce the dose to the next lower dose level;
●否则，保持当前剂量。●Otherwise, maintain current dose.
试验完成后，根据贝叶斯最优区间(BOIN)设计中规定的保序回归选择最接近MTD的剂量。具体而言，选择DLT率的保序估计值最接近目标DLT率的剂量。如果存在相同值，当保序估计值低于目标DLT率时选择较高剂量水平，当保序估计值大于或等于目标DLT率时选择较低剂量水平。After the trial is completed, the dose closest to the MTD is selected based on the ordinal regression specified in the Bayesian optimal interval (BOIN) design. Specifically, the dose whose ordinal estimate of the DLT rate is closest to the target DLT rate is selected. If there is a tie, the higher dose level is selected when the ordinal estimate is lower than the target DLT rate, and the lower dose level is selected when the ordinal estimate is greater than or equal to the target DLT rate.
4.2.3剂量限制性毒性、MTD、MAD和RD的定义4.2.3 Definition of dose-limiting toxicity, MTD, MAD, and RD
适用于第1部分剂量递增：For Part 1 dose escalation:
根据研究者的评估，使用不良事件通用术语标准(CTCAE)第5.0版对所有不良事件进行分级。在DLT评价期间(第1天-第28天)发生的任何以下毒性将被视为DLT，除非与基础恶性肿瘤或外源性原因明确相关。All adverse events were graded using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 based on the investigator's assessment. Any of the following toxicities occurring during the DLT evaluation period (Day 1-Day 28) will be considered a DLT unless clearly related to the underlying malignancy or exogenous cause.
(1)血液学毒性： (1) Hematological toxicity:
1)4级中性粒细胞减少症持续>7天；1) Grade 4 neutropenia lasting for >7 days;
2)3级或4级发热性中性粒细胞减少症；2) Grade 3 or 4 febrile neutropenia;
3)4级贫血；3) Grade 4 anemia;
4)4级血小板减少症持续>7天；4) Grade 4 thrombocytopenia persisting for >7 days;
5)任何持续时间的3级或4级血小板减少伴≥2级出血；5) Grade 3 or 4 thrombocytopenia of any duration accompanied by ≥ Grade 2 bleeding;
6)5级血液学毒性。6) Grade 5 hematological toxicity.
(2)以下任何非血液学毒性：(2) Any of the following non-hematologic toxicities:
1)≥2级免疫性心肌炎；1) ≥ Grade 2 immune myocarditis;
2)≥3级irAE，激素替代治疗可以控制的内分泌毒性除外；2) ≥ Grade 3 irAE, excluding endocrine toxicity that can be controlled by hormone replacement therapy;
3)尽管接受了最佳支持治疗，但任何3级非血液学毒性(非实验室检查值)持续>3天，3) Any grade 3 non-hematologic toxicity (non-laboratory value) persisting for >3 days despite best supportive care,
以下情况除外：The following exceptions apply:
a.最佳支持治疗后持续≤7天的3级恶心；a. Grade 3 nausea that persists for ≤7 days after best supportive care;
b.在≤7天内改善至≤2级的3级疲乏；b. Grade 3 fatigue that improves to ≤ Grade 2 within ≤ 7 days;
c.可通过支持性治疗充分管理或在7天内消退至2级的3级关节痛；c. Grade 3 arthralgia that can be adequately managed with supportive care or resolves to Grade 2 within 7 days;
d.3级皮疹，在接受相当于泼尼松≤10mg/天治疗后7天内消退至≤2级；d. Grade 3 rash that resolves to ≤Grade 2 within 7 days after receiving treatment equivalent to prednisone ≤ 10 mg/day;
e.3级脱发；e. Grade 3 hair loss;
4)任何具有临床意义的3级非血液学实验室检查值异常，如果受试者需要医疗干预治疗，或者该实验室检查异常导致住院，或者该实验室异常持续7天以上。(注意：在给予最佳治疗下，3级AST或ALT升高持续＜3天、3级电解质失衡＜3天，或可控3级高血压将不被视为DLT)。通过甲状腺激素替代治疗控制且受试者无症状的3级甲状腺功能减退将不被视为DLT。4) Any clinically significant grade 3 non-hematological laboratory abnormality, if the subject requires medical intervention, or the laboratory abnormality leads to hospitalization, or the laboratory abnormality persists for more than 7 days. (Note: Grade 3 AST or ALT elevation lasting <3 days, grade 3 electrolyte imbalance <3 days, or controllable grade 3 hypertension under optimal treatment will not be considered a DLT). Grade 3 hypothyroidism controlled by thyroid hormone replacement therapy and the subject is asymptomatic will not be considered a DLT.
5)4级或5级非血液学毒性。5) Grade 4 or 5 non-hematological toxicity.
(3)在DLT评估时间窗内，经申办方/SRC审查后，导致LC-BsAb-002停药或延迟给药>7天的AE也可视为DLT。(3) Within the DLT assessment time window, AEs that lead to discontinuation or delayed administration of LC-BsAb-002 for more than 7 days after review by the sponsor/SRC can also be considered as DLTs.
(4)经申办方与研究者协商审查后，不属于以上DLT标准的具有临床意义或持续存在的AE也可视为DLT。此外，对于给定剂量水平，在DLT评估时间窗后发生的任何具有临床意义的AE(例如，晚期irAE)可视为关于后续剂量水平决策的DLT。(4) After consultation and review between the sponsor and the investigator, clinically significant or persistent AEs that do not meet the above DLT criteria may also be considered DLTs. In addition, for a given dose level, any clinically significant AE (e.g., late irAE) that occurs after the DLT assessment time window may be considered a DLT for subsequent dose level decisions.
最大耐受剂量(MTD)Maximum tolerated dose (MTD)
MTD定义为LC-BsAb-002最高剂量显示真实毒性率<0.3。The MTD was defined as the highest dose of LC-BsAb-002 showing a true toxicity rate < 0.3.
最大给药剂量(MAD)Maximum administered dose (MAD)
MAD定义为在无法确定最大耐受剂量(MTD)的情况下给予的LC-BsAb-002最高剂量。The MAD was defined as the highest dose of LC-BsAb-002 administered when the maximum tolerated dose (MTD) could not be determined.
RD的确定Determination of RD
通过评价第1部分的所有可用PK、PD、疗效、安全性和耐受性数据选择RD。在剂量 爬坡过程中，如果某一特定剂量(已评价DLT)的受试者具有给药相关的疗效反应和/或PK和药效学数据表明药物暴露量在估计的有效暴露范围内，经SRC讨论批准后，可在该剂量水平进行扩展。同时进行PK采样，以进一步评价该剂量水平的安全性、PK和PD特征以及初步疗效，从而帮助确定适当的推荐剂量。The RD was selected by evaluating all available PK, PD, efficacy, safety, and tolerability data from Part 1. During the ramp-up process, if subjects at a specific dose (DLT has been evaluated) have a dosing-related efficacy response and/or PK and pharmacodynamic data indicate that drug exposure is within the estimated effective exposure range, expansion can be carried out at this dose level after discussion and approval by the SRC. PK sampling is also performed to further evaluate the safety, PK and PD characteristics and preliminary efficacy of this dose level, thereby helping to determine the appropriate recommended dose.
以下信息将支持LC-BsAb-002的RD确定：The following information will support the RD determination for LC-BsAb-002:
●MTD或MAD(如果达到)MTD or MAD (if reached)
●PK、PD特征PK and PD characteristics
●活性和安全性的剂量-效应关系●Dose-effect relationship of activity and safety
●至少3例受试者接受该剂量治疗，并在周期1完成所有安全性评估。●At least 3 subjects received this dose and completed all safety assessments in Cycle 1.
这些标准是RD确定的基本组成部分；SRC将做出RD的最终决定。These criteria are an essential component of the RD determination; the SRC will make the final decision on the RD.
4.2.4受试者内剂量递增4.2.4 Intra-subject Dose Escalation
一般而言，本研究不建议进行受试者内剂量递增，但在某些特殊情况下，可根据SRC的建议在符合要求的受试者中进行受试者内剂量递增。In general, within-subject dose escalation is not recommended in this study, but in some special cases, within-subject dose escalation can be performed in eligible subjects based on the recommendations of the SRC.
受试者接受更高剂量的LC-BsAb-002治疗必须符合以下标准：Subjects receiving the higher dose of LC-BsAb-002 must meet the following criteria:
●主管研究者和申办方认可受试者已耐受较低剂量治疗至少4个周期，且未发生任何禁止剂量递增的治疗相关毒性(定义为在最初分配的较低剂量下CTCAE分级≥2级或较基线恶化)。●The subject has tolerated treatment at a lower dose for at least four cycles as agreed by the attending investigator and the sponsor without experiencing any treatment-related toxicity prohibiting dose escalation (defined as CTCAE grade ≥2 or worsening from baseline at the initially assigned lower dose).
●待递增的剂量水平和给药方案必须是已完成DLT评价且已确定对该队列中所有受试者安全且未超过MTD的剂量方案。The dose level and dosing regimen to be escalated must be a dose regimen for which DLT evaluation has been completed and has been determined to be safe for all subjects in the cohort and does not exceed the MTD.
●主管研究者认为这符合受试者的最佳利益。●The responsible investigator believes that this is in the best interests of the subjects.
●需要与申办方协商并达成一致。●Need to negotiate and reach agreement with the applicant.
受试者增加LC-BsAb-002剂量的次数没有限制。对于初始患者内剂量递增后的任何进一步增加，将应用与初始受试者内剂量递增相同的规则。新剂量水平下第1周期的数据将不会正式纳入描述剂量与DLT发生之间关系的统计模型中。但是，该数据将被纳入剂量递增会议的临床安全性评估中。There is no limit to the number of times a subject can increase the dose of LC-BsAb-002. For any further increases after the initial within-patient dose escalation, the same rules as for the initial within-subject dose escalation will apply. Data from cycle 1 at the new dose level will not be formally incorporated into the statistical model describing the relationship between dose and the occurrence of DLTs. However, this data will be included in the clinical safety assessment at the dose escalation meeting.
4.3研究人群4.3 Study population
4.3.1入选标准4.3.1 Inclusion criteria
受试者必须满足以下所有入选标准方可入组本研究：Subjects must meet all of the following inclusion criteria to be included in this study:
1)受试者必须根据监管和机构指南签署书面知情同意书(ICF)并注明日期。这必须在进行任何不被视为标准治疗的方案相关程序之前获得；1) Subjects must sign and date a written informed consent form (ICF) in accordance with regulatory and institutional guidelines. This must be obtained prior to any protocol-related procedure that is not considered standard of care;
2)年龄≥18岁；2) Aged ≥18 years;
3)剂量递增扩展和队列扩展，如有指征，愿意提供肿瘤组织；3) Dose escalation expansion and cohort expansion, willing to provide tumor tissue if indicated;
●如果尽最大努力仍无法获得肿瘤组织，在某些情况下，可在与申办方或指定人员讨论后让受试者入组。(请注意，在获得相关当地卫生部门的批准之前，不会提交肿瘤样本。) If tumor tissue is not available despite best efforts, in certain circumstances, subjects may be enrolled after discussion with the sponsor or designee. (Please note that tumor samples will not be submitted until approval has been obtained from the relevant local health authorities.)
4)受试者需满足对应入组研究部分的相应要求：4) Subjects must meet the corresponding requirements of the corresponding study section:
第1部分：part 1:
剂量递增及剂量递增扩展：受试者的组织学或细胞学诊断必须为不适合局部治疗的任何类型的转移性或局部晚期实体瘤；受试者必须标准治疗失败或不适合标准治疗。Dose escalation and dose expansion: Subjects must have a histological or cytological diagnosis of any type of metastatic or locally advanced solid tumor that is not suitable for local treatment; subjects must have failed standard treatment or are not suitable for standard treatment.
第2部分：part 2:
将入组既往接受过包括PD1/PDL1抑制剂在内的癌症免疫治疗(CIT)的受试者。队列扩展人群的选择将基于最新数据，如第1部分研究的结果和/或具有相同/相似作用机制的其他药物的累积新数据，包括但不限于以下瘤种：Subjects who have previously received cancer immunotherapy (CIT) including PD1/PDL1 inhibitors will be enrolled. The selection of the cohort expansion population will be based on the latest data, such as the results of the Part 1 study and/or the accumulated new data of other drugs with the same/similar mechanism of action, including but not limited to the following tumor types:
●NSCLC队列：经组织学或细胞学证实的不可治愈的晚期NSCLC受试者；对于无驱动基因突变的受试者，必须接受至少1种但不超过2种系统抗肿瘤治疗后进展；对于有驱动基因突变的受试者，必须接受至少1种已确定的标准抗肿瘤靶向治疗失败，或根据研究者的判断不适合目前的标准治疗。(辅助治疗完成后6个月内进展被视为一线治疗失败。)●NSCLC cohort: subjects with incurable advanced NSCLC confirmed by histology or cytology; subjects without driver gene mutations must have progressed after at least 1 but no more than 2 systemic anti-tumor therapies; subjects with driver gene mutations must have failed at least 1 established standard anti-tumor targeted therapy, or be unsuitable for current standard treatment based on the investigator's judgment. (Progression within 6 months after completion of adjuvant therapy is considered failure of first-line treatment.)
●HCC队列：经组织学或细胞学证实的HCC(排除已知的纤维板层HCC或混合性胆管癌和HCC)；Child-Pugh评分≤7，无肝性脑病；巴塞罗那临床肝癌(BCLC)分期为B或C期，不适合手术或局部治疗，或在手术和/或局部治疗后进展；至少1种含抗血管生成疗法的治疗失败，但不超过2种全身抗肿瘤治疗的受试者。●HCC cohort: HCC confirmed by histology or cytology (excluding known fibrolamellar HCC or mixed cholangiocarcinoma and HCC); Child-Pugh score ≤7, no hepatic encephalopathy; Barcelona Clinic Liver Cancer (BCLC) stage B or C, not suitable for surgery or local treatment, or progressing after surgery and/or local treatment; subjects who have failed at least 1 anti-angiogenic therapy but no more than 2 systemic anti-tumor therapies.
●其他实体瘤队列：经组织学或细胞学证实的非NSCLC和HCC的恶性实体瘤。在接受至少1种标准治疗后发生进展的受试者。●Other solid tumor cohort: Subjects with histologically or cytologically confirmed malignant solid tumors other than NSCLC and HCC who have progressed after receiving at least 1 standard treatment.
5)根据RECIST 1.1版标准，至少存在一个可测量病灶。既往接受过局部治疗或局部区域治疗(如放疗、肝动脉栓塞、射频消融和经皮介入治疗)的病灶不应视为可测量病灶，除非在局部治疗或局部区域治疗后观察到进展；5) According to the RECIST version 1.1 standard, there is at least one measurable lesion. Lesions that have previously received local or locoregional treatment (such as radiotherapy, hepatic artery embolization, radiofrequency ablation, and percutaneous intervention) should not be considered measurable lesions unless progression is observed after local or locoregional treatment;
6)ECOG体能状态评分为0或1；6) ECOG performance status score of 0 or 1;
7)预期寿命≥12周；7) Life expectancy ≥ 12 weeks;
8)筛选期实验室检查值必须符合以下标准(在研究治疗首次给药前2周内接受过输血、血液成分输注或集落刺激因子(例如，粒细胞集落刺激因子[G-CSF]、粒细胞巨噬细胞集落刺激因子[GM-CSF]或重组促红细胞生成素等)的受试者除外)：8) Laboratory test values during the screening period must meet the following criteria (except for subjects who have received blood transfusion, blood component transfusion, or colony stimulating factors (e.g., granulocyte colony stimulating factor [G-CSF], granulocyte macrophage colony stimulating factor [GM-CSF], or recombinant erythropoietin, etc.) within 2 weeks before the first dose of study treatment):
●血液学：中性粒细胞绝对计数(ANC)≥1.5×10 9/L；血小板≥100×10 9/L，血红蛋白≥90g/L。●Hematology: absolute neutrophil count (ANC) ≥1.5×10 9/L; platelet ≥100×10 9/L, hemoglobin ≥90g/L.
●肝脏：ALT和AST≤2.5×正常值上限(ULN)，若伴肝转移或肝癌受试者ALT和AST≤5.0×ULN；血清总胆红素(T-BIL)≤1.5×ULN，若伴肝转移或肝癌受试者T-BIL≤2.0×ULN。●Liver: ALT and AST ≤ 2.5 × upper limit of normal (ULN); if the subject has liver metastasis or liver cancer, ALT and AST ≤ 5.0 × ULN; serum total bilirubin (T-BIL) ≤ 1.5 × ULN; if the subject has liver metastasis or liver cancer, T-BIL ≤ 2.0 × ULN.
●肾脏：血清肌酐(Cr)≤1.5×ULN，或Cr清除率≥60.0mL/min(使用Cockcroft-Gault公式计算或测量)。●Kidney: Serum creatinine (Cr) ≤1.5×ULN, or Cr clearance ≥60.0 mL/min (calculated using the Cockcroft-Gault formula or measured).
Cockcroft-Gault公式： Cockcroft-Gault formula:
男性：Cr清除率＝((140-年龄)×体重)/(72×血清Cr)Male: Cr clearance = ((140-age) × body weight) / (72 × serum Cr)
女性：Cr清除率＝((140-年龄)×体重)/(72×血清Cr)×0.85Female: Cr clearance = ((140-age) × body weight) / (72 × serum Cr) × 0.85
体重单位：kg；血清Cr单位：mg/dLBody weight unit: kg; serum Cr unit: mg/dL
●凝血：凝血：国际标准化比值(INR)≤1.5或凝血活酶时间(PT)≤1.5×ULN(对于接受抗凝治疗的受试者，PT和INR必须在抗凝剂预期用途的治疗范围内)。● Coagulation: Coagulation: International normalized ratio (INR) ≤ 1.5 or thromboplastin time (PT) ≤ 1.5 × ULN (for subjects receiving anticoagulant therapy, PT and INR must be within the therapeutic range for the intended use of the anticoagulant).
●心脏功能良好：左心室射血分数(LVEF)>50％。●Good heart function: left ventricular ejection fraction (LVEF)>50%.
9)有生育能力的女性(WOCBP)必须同意在整个研究治疗期间和研究治疗末次给药后180天内使用可接受的避孕方法，以尽量减少妊娠风险。9) Women of childbearing potential (WOCBP) must agree to use acceptable contraceptive methods throughout the study treatment period and within 180 days after the last dose of study treatment to minimize the risk of pregnancy.
10)WOCBP在试验用药品开始给药前72小时内的血清或尿妊娠试验结果必须为阴性(最低灵敏度为25IU/L或等效单位的HCG)。10) WOCBP must have a negative serum or urine pregnancy test result (minimum sensitivity of 25 IU/L or equivalent units of HCG) within 72 hours before the start of administration of the investigational drug.
11)女性不得处于哺乳期。11) Women must not be breastfeeding.
12)男性受试者必须同意从研究治疗首次给药开始至研究治疗末次给药后180天内采取可接受的避孕措施。12) Male subjects must agree to take acceptable contraceptive measures from the first dose of study treatment to 180 days after the last dose of study treatment.
4.3.2排除标准4.3.2 Exclusion criteria
符合以下任何标准的受试者无资格参加本研究：Subjects who meet any of the following criteria are not eligible to participate in this study:
1)受试者在研究治疗首次给药前5个半衰期或2周内(以较短者为准)接受过细胞毒治疗、抗肿瘤靶向小分子(例如酪氨酸激酶抑制剂)、激素药物；在研究治疗首次给药前5个半衰期或4周内(研究第1部分剂量递增部分以较长者为准；研究第1部分剂量递增扩展及研究第2部分以较短者为准)接受过单克隆抗体(mAb)；在研究治疗首次给药前2周内接受过抗肿瘤中药制剂或接受过任何放疗。1) Subjects have received cytotoxic therapy, anti-tumor targeted small molecules (such as tyrosine kinase inhibitors), or hormonal drugs within 5 half-lives or 2 weeks (whichever is shorter) before the first dose of study treatment; received monoclonal antibodies (mAb) within 5 half-lives or 4 weeks (the longer of the dose escalation part of Part 1 of the study; the shorter of the dose escalation extension of Part 1 and Part 2 of the study) before the first dose of study treatment; received anti-tumor Chinese medicine preparations or any radiotherapy within 2 weeks before the first dose of study treatment.
2)受试者目前正在参与并接受研究治疗，或在研究治疗首次给药前4周内参与过试验用药物的研究并接受过研究治疗或使用过试验用器械，注：这不包括研究随访期；2) The subject is currently participating in and receiving study treatment, or has participated in a study of an investigational drug and received study treatment or used an investigational device within 4 weeks before the first dose of study treatment. Note: This does not include the study follow-up period;
3)既往接受过其他TIGIT或PVRIG抗体治疗；本方案中未明确描述的既往癌症免疫治疗应与医学监查员讨论，以确定潜在合格性。3) Previous treatment with other TIGIT or PVRIG antibodies; previous cancer immunotherapy not clearly described in this protocol should be discussed with the medical monitor to determine potential eligibility.
4)研究治疗首次给药前2年内患有任何其他恶性肿瘤，但认为已治愈且研究者认为复发风险较低的局部癌症除外，如包括基底或鳞状细胞皮肤癌、浅表性膀胱癌或前列腺、子宫颈或乳腺原位癌；4) Suffering from any other malignant tumor within 2 years before the first dose of study treatment, except for local cancers that are considered to be cured and the investigator believes that the risk of recurrence is low, such as basal or squamous cell skin cancer, superficial bladder cancer, or prostate, cervical or breast carcinoma in situ;
5)受试者尚未从既往抗肿瘤治疗导致的AE中恢复(即，恢复至≤1级或基线水平)。注：不影响受试者安全性的≤2级AE除外，可有资格参加研究，例如化疗引起的≤2级脱发和神经病变。5) Subjects have not recovered from AEs caused by previous anti-tumor treatment (i.e., recovered to ≤ Grade 1 or baseline level). Note: Subjects with ≤ Grade 2 AEs that do not affect the safety of the subject are eligible to participate in the study, such as ≤ Grade 2 alopecia and neuropathy caused by chemotherapy.
6)近期(研究治疗首次给药前2年内)有活动性憩室炎或症状性消化性溃疡病史的受试者；6) Subjects with a recent history of active diverticulitis or symptomatic peptic ulcer (within 2 years before the first dose of study treatment);
7)研究治疗首次给药前4周内接受过大手术或1周内接受过开放性活检(不包括穿刺)； 7) Major surgery within 4 weeks or open biopsy (excluding puncture) within 1 week before the first dose of study treatment;
8)存在需要立即放疗或类固醇治疗的中枢神经系统(CNS)转移/软脑膜疾病(CNS转移得到控制的受试者可参加本试验，前提是其在进入研究前临床稳定至少4周，无新发脑转移或脑转移扩大的证据，并且在接受研究治疗首次给药前已停用类固醇至少2周)；8) Central nervous system (CNS) metastases/leptomeningeal disease requiring immediate radiotherapy or steroid treatment (subjects with controlled CNS metastases may participate in this trial, provided that they are clinically stable for at least 4 weeks before entering the study, have no evidence of new brain metastases or brain metastases expansion, and have stopped taking steroids for at least 2 weeks before the first dose of study treatment);
9)受试者在1年内有活动性肺结核感染史；有活动性肺结核病史的受试者，如果研究者判断在研究治疗首次给药前超过1年没有活动性肺结核的证据，则适合入组；9) Subjects with a history of active tuberculosis infection within 1 year; Subjects with a history of active tuberculosis are suitable for inclusion if the investigator determines that there is no evidence of active tuberculosis more than 1 year before the first dose of study treatment;
10)受试者在研究治疗首次给药前6个月内患有具有临床意义的心血管疾病，包括但不限于心肌梗死、重度/不稳定型心绞痛、原发性心肌病、脑血管意外(包括短暂性脑缺血、脑出血、脑梗死)或充血性心力衰竭(纽约心脏病协会心功能分级>2级)；需要药物治疗的症状性冠心病；需要药物治疗的心律失常；心电图显示QTcF间期>480ms；或未控制的高血压(充分药物治疗后收缩压≥160mmHg和/或舒张压≥100mmHg)；10) Subjects with clinically significant cardiovascular disease within 6 months before the first administration of study treatment, including but not limited to myocardial infarction, severe/unstable angina, primary cardiomyopathy, cerebrovascular accident (including transient ischemic attack, cerebral hemorrhage, cerebral infarction) or congestive heart failure (New York Heart Association functional grade>2); symptomatic coronary heart disease requiring drug treatment; arrhythmia requiring drug treatment; electrocardiogram showing QTcF interval>480ms; or uncontrolled hypertension (systolic blood pressure ≥160mmHg and/or diastolic blood pressure ≥100mmHg after adequate drug treatment);
11)研究药物首次给药前4周内出现需要引流的腹水、胸腔积液或心包积液；11) Ascites, pleural effusion or pericardial effusion requiring drainage occurred within 4 weeks before the first administration of the study drug;
12)已知有免疫缺陷病毒(HIV)感染史或已知有获得性免疫缺陷综合征(AIDS)；12) Known history of HIV infection or acquired immunodeficiency syndrome (AIDS);
13)活动性或慢性乙型肝炎(HBsAg或HBcAb阳性且HBV DNA≥2000IU/mL或≥10000拷贝/mL)或丙型肝炎(HCV抗体阳性且HCV RNA≥ULN)感染受试者；筛选时HBsAg阳性或NBV-DNA检测阳性的受试者建议在研究期间根据当地实践接受抗病毒治疗；13) Subjects with active or chronic hepatitis B (HBsAg or HBcAb positive and HBV DNA ≥ 2000 IU/mL or ≥ 10000 copies/mL) or hepatitis C (HCV antibody positive and HCV RNA ≥ ULN); Subjects with HBsAg positive or NBV-DNA positive test at screening are recommended to receive antiviral treatment according to local practice during the study;
14)在研究药物给药前14天内接受任何其他抗肿瘤治疗或全身性免疫抑制药物(包括但不限于泼尼松>10mg/天或等效药物)或全身性皮质类固醇治疗。14) Receiving any other anti-tumor treatment or systemic immunosuppressive drugs (including but not limited to prednisone >10 mg/day or equivalent) or systemic corticosteroid treatment within 14 days before administration of study drug.
注：如果无活动性自身免疫性疾病，允许使用>10mg泼尼松或等效药物进行吸入性或局部类固醇或肾上腺替代治疗。允许短期(7天)使用类固醇预防(例如，用于造影剂过敏)或治疗非自身免疫性疾病(例如，造影剂过敏引起的迟发型超敏反应)；Note: Inhaled or topical steroids or adrenal replacement therapy with >10 mg prednisone or equivalent are permitted if there is no active autoimmune disease. Short-term (7 days) use of steroids for prophylaxis (e.g., for contrast allergy) or treatment of non-autoimmune disease (e.g., delayed hypersensitivity reactions due to contrast allergy) is permitted;
15)已知或疑似活动性自身免疫性疾病。可纳入患有白癜风、仅需要激素替代治疗的残留甲状腺功能减退症、不需要全身治疗的银屑病或在无外部触发因素的情况下预期不会复发的疾病、1型糖尿病(可通过胰岛素治疗控制血糖)的受试者。既往暴露于抗PD-1/PD-L1治疗且已充分治疗的皮疹或接受内分泌疾病替代治疗的受试者可入选。15) Known or suspected active autoimmune disease. Subjects with vitiligo, residual hypothyroidism requiring only hormone replacement therapy, psoriasis that does not require systemic treatment or diseases that are not expected to relapse without external triggers, and type 1 diabetes (blood sugar can be controlled by insulin therapy) can be included. Subjects with a rash that has been previously exposed to anti-PD-1/PD-L1 therapy and has been adequately treated or receiving replacement therapy for endocrine diseases can be included.
16)有间质性肺炎病史、活动性肺炎证据(允许有放射野放射性肺炎[纤维化]史)和研究者认为不合适的活动性肺炎；16) A history of interstitial pneumonia, evidence of active pneumonia (a history of radiation pneumonitis [fibrosis] in the radiation field is allowed), and active pneumonia that the investigator considers inappropriate;
17)对嵌合或人源化抗体或融合蛋白有严重超敏反应史；17) History of severe hypersensitivity reaction to chimeric or humanized antibodies or fusion proteins;
18)有同种异体组织/实体器官移植或移植物抗宿主病病史；18) History of allogeneic tissue/solid organ transplantation or graft-versus-host disease;
19)已知的活动性感染，需要在研究治疗首次给药前2周内通过静脉输注进行全身治疗；19) Known active infection requiring systemic treatment by intravenous infusion within 2 weeks before the first dose of study treatment;
20)在研究治疗首次给药前4周内使用任何活性疫苗治疗；20) Use of any active vaccine within 4 weeks before the first dose of study treatment;
21)已知会干扰试验要求的精神疾病或药物滥用； 21) Mental illness or drug abuse that is known to interfere with trial requirements;
22)存在研究者认为会使研究药物给药对受试者造成危害或使毒性判定或不良事件的解释变得模糊的基础疾病；22) The presence of an underlying disease that the investigator believes will cause harm to the subjects by administering the study drug or will obscure the determination of toxicity or the interpretation of adverse events;
23)研究人员认为不适合入选的其他情况。23) Other situations that the researchers consider inappropriate for inclusion.
4.3.3筛选失败4.3.3 Screening failure
签署ICF但被认为不合格的受试者将被视为筛选失败。筛选失败的原因将录入电子病例报告表(eCRF)。签署ICF后，将为每例受试者分配一个筛选编号。筛选失败是指受试者被分配参加研究但未接受治疗。应提供筛选失败受试者的下列信息：Subjects who sign the ICF but are deemed ineligible will be considered screen failures. The reason for screen failure will be recorded in the electronic case report form (eCRF). After signing the ICF, each subject will be assigned a screening number. Screen failure is when a subject was assigned to the study but did not receive treatment. The following information should be provided for subjects who failed the screen:
●访视日期Visit date
●筛选编号●Screening number
●人口统计学，包括当地法规允许的人种/种族、年龄和性别● Demographics, including race/ethnicity, age, and gender as permitted by local regulations
●筛选失败的原因●Reasons for screening failure
●严重不良事件信息(如有)●Serious adverse event information (if any)
如果在筛选期内有重复检查结果，则应使用最接近估计给药日期的结果进行入选/排除标准评估。对于筛选失败的受试者，研究者应在与申办方的医学监查员沟通后确定受试者是否可以重新筛选。If there are duplicate test results during the screening period, the results closest to the estimated dosing date should be used for inclusion/exclusion criteria assessment. For subjects who fail the screening, the investigator should determine whether the subject can be rescreened after communicating with the sponsor's medical monitor.
将由申办方或指定的合同研究组织(CRO)协调在第1部分和第2部分研究期间分配受试者至特定队列。The assignment of subjects to specific cohorts during Parts 1 and 2 of the study will be coordinated by the sponsor or a designated contract research organization (CRO).
对于不符合入选标准或符合排除标准但错误接受研究治疗的受试者，研究者应与申办方讨论并确定受试者是否可以继续研究治疗。For subjects who do not meet the inclusion criteria or meet the exclusion criteria but receive study treatment in error, the investigator should discuss with the sponsor and determine whether the subject can continue with the study treatment.
4.4研究治疗4.4 Study Treatment
4.4.1研究药物4.4.1 Study Drugs
抗TIGIT/抗PVRIG抗体LC-BsAb-002(PCT/CN2022/108648)注射液。活性成分为：抗TIGIT/抗PVRIG抗体，50mg/mL。辅料为：醋酸-醋酸钠，pH5.0；蔗糖；PS80。Anti-TIGIT/anti-PVRIG antibody LC-BsAb-002 (PCT/CN2022/108648) injection. Active ingredient: anti-TIGIT/anti-PVRIG antibody, 50 mg/mL. Excipients: acetic acid-sodium acetate, pH 5.0; sucrose; PS80.
规格：300mg/瓶Specification: 300mg/bottle
储存条件：2-8℃Storage conditions: 2-8℃
4.4.2给药方案4.4.2 Dosage regimen
抗TIGIT/抗PVRIG抗体通过静脉输注给药。剂量递增计划为10、30、100、300和600mg，采用半对数递增策略，但最后2个剂量组除外，采用100％递增。从第一周期开始，每28天为1周期，QW给药，直至疾病进展、出现不可耐受的毒性、撤回知情同意或死亡。为了降低输液反应的风险，所有受试者的首次输注应在不少于90min时间内完成。根据输液相关操作调整输液速度，确保受试者安全。如果未观察到输液反应，后续输注时间不少于60min。Anti-TIGIT/anti-PVRIG antibodies are administered by intravenous infusion. The dose escalation plan is 10, 30, 100, 300 and 600 mg, using a semi-logarithmic escalation strategy, except for the last 2 dose groups, which use 100% escalation. Starting from the first cycle, every 28 days is 1 cycle, and QW administration is continued until disease progression, intolerable toxicity, withdrawal of informed consent or death. To reduce the risk of infusion reactions, the first infusion of all subjects should be completed in no less than 90 minutes. Adjust the infusion rate according to infusion-related operations to ensure the safety of the subjects. If no infusion reaction is observed, the subsequent infusion time shall be no less than 60 minutes.
其他不超过MTD的剂量水平，如中间剂量水平或高于600mg QW的剂量或替代给药方案可根据安全性、耐受性和药代动力学或根据申办方和PI在第1部分期间通过SRC共同决定进行评价。Other dose levels not exceeding the MTD, such as intermediate dose levels or doses above 600 mg QW or alternative dosing regimens may be evaluated based on safety, tolerability, and pharmacokinetics or as jointly determined by the sponsor and PI through the SRC during Part 1.
如果在治疗期间因不良事件中断抗TIGIT/抗PVRIG抗体的计划给药，可暂停或延迟 研究药物给药，直至受试者符合重新开始研究药物给药的标准。If the planned dosing of anti-TIGIT/anti-PVRIG antibodies is interrupted due to adverse events during treatment, it may be suspended or delayed. Study drug was administered until the subject met the criteria for restarting study drug administration.
受试者将继续接受研究治疗，直至出现不可耐受的毒性，或直至研究者综合评价影像学和实验室检查数据以及受试者的临床状况(例如，肿瘤症状恶化)后确定受试者缺乏临床获益，或直至受试者自愿退出研究治疗，以先发生者为准。Subjects will continue to receive study treatment until intolerable toxicity occurs, or until the investigator determines that the subject lacks clinical benefit after comprehensive evaluation of imaging and laboratory test data and the subject's clinical condition (e.g., worsening of tumor symptoms), or until the subject voluntarily withdraws from study treatment, whichever occurs first.
4.4.3剂量调整4.4.3 Dosage Adjustment
根据CTCAE v5.0评估不良事件的严重程度，可参考以下指南调整LC-BsAb-002的剂量。The severity of adverse events was assessed according to CTCAE v5.0 and the dose of LC-BsAb-002 can be adjusted according to the following guidelines.
1.除非受试者可能从研究者评估和申办方批准的治疗中获益，否则不会降低LC-BsAb-002的剂量。1. The dose of LC-BsAb-002 will not be reduced unless the subject is likely to benefit from treatment as assessed by the investigator and approved by the sponsor.
2.在第2部分中，随着安全性数据的积累，如果SRC选择了可能的新的RD，具体情况根据研究者判断，可在无疾病进展的受试者中进行受试者内剂量调整。2. In Part 2, as safety data accumulate, if the SRC selects a possible new RD, within-subject dose adjustments may be made in subjects without disease progression based on the investigator's judgment.
3.治疗期间不良事件(TEAE)均应视为与研究治疗相关，并可能需要调整剂量，除非与外源性原因明确相关。3. All treatment-emergent adverse events (TEAEs) should be considered related to the study treatment and may require dose adjustment unless clearly related to exogenous causes.
4.如果暂停LC-BsAb-002给药，则在TEAE消退至≤1级或基线值(或2级，研究者认为对受试者无安全性风险)后可重新开始LC-BsAb-002给药。4. If LC-BsAb-002 administration is suspended, LC-BsAb-002 administration may be restarted after the TEAE resolves to ≤ Grade 1 or baseline value (or Grade 2, if the investigator determines that there is no safety risk to the subject).
5.如果因TEAE(未评估为irAE)导致给药延迟超过4周，则应永久停用LC-BsAb-002，除非与申办方另有讨论。5. If dosing is delayed for more than 4 weeks due to a TEAE (not assessed as an irAE), LC-BsAb-002 should be permanently discontinued unless otherwise discussed with the sponsor.
6.第2周期治疗的给药时间窗为±3天。LC-BsAb-002需要在窗口期给药。如果错过给药窗口，LC-BsAb-002将跳过本次给药并在下一次计划访视时给药。6. The dosing window for the second cycle of treatment is ±3 days. LC-BsAb-002 needs to be administered during the window period. If the dosing window is missed, LC-BsAb-002 will skip this administration and be administered at the next scheduled visit.
7.对于irAE7. For irAE
●当irAE消退至≤1级时，应开始类固醇减量，并持续不少于4周。●When the irAE resolves to ≤ grade 1, steroid tapering should be initiated and continued for at least 4 weeks.
●如果皮质类固醇在12周内不能减量至≤10mg/天泼尼松或等效药物，则应永久停用LC-BsAb-002。●If corticosteroids cannot be tapered to ≤10 mg/day prednisone or equivalent within 12 weeks, LC-BsAb-002 should be permanently discontinued.
●对于重度和危及生命的irAE，应首先开始静脉注射糖皮质激素治疗，然后口服类固醇。如果皮质类固醇无法控制irAE，则应开始其他免疫抑制治疗。●For severe and life-threatening irAEs, treatment with intravenous glucocorticoids should be started first, followed by oral steroids. If the irAE cannot be controlled by corticosteroids, other immunosuppressive therapy should be started.
LC-BsAb-002可能因治疗相关AE以外的情况而中断给药，如与研究治疗无关的内科/外科事件或行政原因。在这种情况下，除非与申办方另有讨论，受试者应在末次给药后4周内重新接受研究治疗。中断给药的原因应记录在患者的研究记录中。LC-BsAb-002 dosing may be interrupted for circumstances other than treatment-related AEs, such as medical/surgical events unrelated to study treatment or administrative reasons. In such cases, subjects should be restarted on study treatment within 4 weeks of the last dose unless otherwise discussed with the sponsor. The reason for interruption should be recorded in the patient's study record.
如果受试者在LC-BsAb-002再次使用后再次发生相同级别或相同级别的重度或危及生命的事件，则应终止试验治疗。推荐的剂量调整见表9。If a subject experiences a recurrence of a severe or life-threatening event of the same grade or higher after re-administration of LC-BsAb-002, trial treatment should be discontinued. Recommended dose adjustments are shown in Table 9.
表9治疗中出现的不良事件的剂量调整指南

Table 9 Dose adjustment guidelines for treatment-emergent adverse events

4.5安全性评价 4.5 Safety evaluation
将采用NCI-CTCAE v5.0标准进行安全性评价。The NCI-CTCAE v5.0 standard will be used for safety evaluation.
在研究期间(受试者签署知情同意书至完成安全性随访或退出研究)，观察并记录所有受试者在研究过程中出现的任何不良事件，包括但不限于异常临床症状、生命体征、实验室检查异常等，并评估其与研究药物的相关性。During the study (from the time the subject signs the informed consent to the completion of safety follow-up or withdrawal from the study), observe and record any adverse events that occur in all subjects during the study, including but not limited to abnormal clinical symptoms, vital signs, abnormal laboratory tests, etc., and evaluate their relevance to the study drug.
4.5.1临床实验室检查4.5.1 Clinical laboratory tests
实验室参数将在当地研究中心进行检测。各研究中心检测的参数总结如下：Laboratory parameters will be tested at local research centers. The parameters tested at each research center are summarized as follows:
●血液学检查：WBC、ANC、LYM、MONO、EOS、BASO、RBC、HB、PLT。●Hematological examination: WBC, ANC, LYM, MONO, EOS, BASO, RBC, HB, PLT.
●尿液分析：pH、UWBC、尿蛋白、URBC。●Urinalysis: pH, UWBC, urine protein, URBC.
●血生化：TBIL、DBIL、ALT、AST、ALP、GGT、TP、ALB、尿素或BUN、Cr、UA、Glu、K、Na、Cl、Ca、Mg、P、LIP、AMY、LDL、HDL、LDH。●Blood biochemistry: TBIL, DBIL, ALT, AST, ALP, GGT, TP, ALB, urea or BUN, Cr, UA, Glu, K, Na, Cl, Ca, Mg, P, LIP, AMY, LDL, HDL, LDH.
●心肌酶：CK、CK-MB；cTnT或cTnI(如需要)。Cardiac enzymes: CK, CK-MB; cTnT or cTnI (if necessary).
●血清HCG/尿妊娠试验。●Serum HCG/urine pregnancy test.
●凝血：INR或PT。●Coagulation: INR or PT.
●粪便潜血试验。●Fecal occult blood test.
●甲状腺功能检查：FT3或T3、FT4或T4、TSH。●Thyroid function tests: FT3 or T3, FT4 or T4, TSH.
●病毒学检查：HIV、HBsAg、HBsAb、HBcAb、HCV-Ab；定量HCV-RNA和定量HBV-DNA(如需要)。●Virological examination: HIV, HBsAg, HBsAb, HBcAb, HCV-Ab; quantitative HCV-RNA and quantitative HBV-DNA (if necessary).
●AFP(仅HCC)AFP (HCC only)
上述检查的流程和时间点见评估时间表。如有临床指征，可进行额外的实验室检查。The procedures and timing of the above tests are shown in the Assessment Schedule. Additional laboratory tests may be performed if clinically indicated.
实验室检查结果必须由研究者或有资质的人员审查和确认。研究期间发生的具有临床意义的实验室检查异常必须记录在CRF的不良事件部分。Laboratory test results must be reviewed and confirmed by the investigator or qualified personnel. Clinically significant laboratory test abnormalities occurring during the study must be recorded in the adverse events section of the CRF.
4.5.2体格检查4.5.2 Physical examination
研究者应对受试者进行全面的体格检查，包括身高、体重、一般状况、皮肤/粘膜、五官、头颈部、肺/胸部、腹部、脊柱/四肢、神经系统和泌尿生殖系统。身高仅需要在筛选期间进行测量。研究者可根据症状进行特定的体格检查。The investigator should conduct a comprehensive physical examination of the subject, including height, weight, general condition, skin/mucous membranes, facial features, head and neck, lungs/chest, abdomen, spine/limbs, nervous system, and urogenital system. Height only needs to be measured during the screening period. The investigator may conduct specific physical examinations based on symptoms.
4.5.3生命体征4.5.3 Vital signs
研究者或研究者授权的其他研究人员应根据方案规定确定受试者的生命体征。生命体征包括体温、心率、呼吸频率和血压。The investigator or other researchers authorized by the investigator should determine the subject's vital signs according to the protocol. Vital signs include body temperature, heart rate, respiratory rate and blood pressure.
4.5.4 12导联心电图(ECG)4.5.4 12-lead electrocardiogram (ECG)
应在研究中心使用标准流程进行标准12导联ECG检查，包括测量心率和QTcF间期。评估时间表中规定了ECG测量的时间点。如有临床指征，可进行额外的ECG检查。A standard 12-lead ECG, including measurement of heart rate and QTcF interval, should be performed at the site using standard protocols. The timing of ECG measurements is specified in the evaluation schedule. Additional ECGs may be performed as clinically indicated.
4.5.5超声心动图4.5.5 Echocardiography
受试者将在筛选期间接受超声心动图检查，并记录LVEF。根据研究治疗期间的ECG结果，研究者可酌情决定进行额外的超声心动图检查。 Subjects will undergo an echocardiogram during the screening period and LVEF will be recorded. Additional echocardiograms may be performed at the discretion of the investigator based on ECG results during study treatment.
4.6药代动力学和免疫原性评价4.6 Pharmacokinetic and immunogenicity evaluation
第1部分PK和免疫原性样本采集：Part 1 PK and Immunogenicity Sample Collection:
将采集血清样本用于测定LC-BsAb-002的药代动力学和免疫原性。将通过直接静脉穿刺或在前臂静脉插入留置插管采集血样，从输注部位对侧手臂采集样本，每次采集4mL静脉血用于PK检测，以及4.5mL静脉血用于免疫原性检测(3mL用于ADA检测和备份，1.5mL用于Nab检测)。对于所有PK和免疫原性((ADA/Nab))样本，必须在适当的eCRF中记录给药的确切日期和时间，以及血液样本的实际采集日期和时间。有关血样采集、处理、储存和运输的详细信息，请参见中心实验室手册。Serum samples will be collected for determination of the pharmacokinetics and immunogenicity of LC-BsAb-002. Blood samples will be collected by direct venipuncture or by insertion of an indwelling cannula in the forearm vein, with samples collected from the arm contralateral to the infusion site, with 4 mL of venous blood collected each time for PK testing and 4.5 mL of venous blood collected for immunogenicity testing (3 mL for ADA testing and backup, 1.5 mL for Nab testing). For all PK and immunogenicity ((ADA/Nab)) samples, the exact date and time of administration, as well as the actual date and time of blood sample collection, must be recorded in the appropriate eCRF. Please refer to the central laboratory manual for detailed information on blood sample collection, handling, storage, and transportation.
第2部分PK和免疫原性样本采集：Part 2 PK and Immunogenicity Sample Collection:
第2部分的PK和免疫原性(ADA/Nab)采样策略可根据第1部分的临床药代动力学特征实测结果或用药方案变更进行调整优化。The PK and immunogenicity (ADA/Nab) sampling strategy of Part 2 can be adjusted and optimized based on the actual measured results of the clinical pharmacokinetic characteristics of Part 1 or changes in the medication regimen.
分析方法：将采用经验证的分析方法对LC-BsAb-002的PK和免疫原性样本进行生物分析。 Analytical Methods: Bioanalysis of PK and immunogenicity samples of LC-BsAb-002 will be performed using validated analytical methods.
4.7实体瘤疗效评价4.7 Evaluation of efficacy in solid tumors
将根据RECIST 1.1版标准对实体瘤靶病灶和非靶病灶进行疗效评估。The efficacy of solid tumor target lesions and non-target lesions will be evaluated according to the RECIST version 1.1 criteria.
应使用增强CT或MRI获得肿瘤影像。扫描区域应包括胸部、腹部、骨盆及脑部。其他区域是否需要扫描将由研究者根据具体情况决定。同一受试者在整个试验过程中的成像方法应保持一致(包括成像方法、范围、模式、造影剂等)。Enhanced CT or MRI should be used to obtain tumor images. The scan area should include the chest, abdomen, pelvis and brain. Whether other areas need to be scanned will be determined by the investigator based on specific circumstances. The imaging method for the same subject should be consistent throughout the trial (including imaging method, range, mode, contrast agent, etc.).
将在首次给药前4周进行肿瘤的基线影像学评估。如果肿瘤评估是在签署知情同意书之前进行的，并且是在首次给药前4周内进行的，在获得申办方同意后，可以用该结果替代基线影像学评估。筛选时，研究者将根据RECIST 1.1版标准确认靶病灶和基线影像。Baseline imaging assessment of the tumor will be performed 4 weeks before the first dose. If the tumor assessment is performed before the signing of the informed consent form and is performed within 4 weeks before the first dose, this result can be used in place of the baseline imaging assessment with the consent of the sponsor. At screening, the investigator will confirm the target lesions and baseline imaging according to the RECIST version 1.1 criteria.
首次给药后每8±1周进行一次影像学评估。研究者可根据受试者的实际情况进行计划外影像学评估。影像学评估的时间点应遵循日历日，不应因治疗延迟或计划外影像学评估而改变。影像学评估应持续至开始新的抗肿瘤治疗、疾病进展、退出研究或死亡，以先发生者为准。如果在距离下一个计划影像检查时间点不超过4周的时间内进行了计划外影像评估，则可以不进行下一次计划成像评估，可在后续计划成像时间点继续进行成像评估。Imaging assessments will be performed every 8±1 weeks after the first dose. Investigators may perform unplanned imaging assessments based on the actual situation of the subjects. The timing of imaging assessments should follow calendar days and should not be changed due to treatment delays or unplanned imaging assessments. Imaging assessments should continue until the start of new anti-tumor treatment, disease progression, withdrawal from the study, or death, whichever occurs first. If an unplanned imaging assessment is performed within 4 weeks of the next scheduled imaging examination time point, the next scheduled imaging assessment may not be performed, and imaging assessments may continue at subsequent scheduled imaging time points.
根据RECIST 1.1版，肿瘤疗效评估分为CR(完全缓解)、PR(部分缓解)、SD(疾病稳定)和PD(疾病进展)。对于CR或PR受试者，应在首次记录PR或CR日期后≥4周确认缓解。对于评估为PD的受试者，如果研究者和申办方认为受试者可能从继续治疗中获益，则可继续治疗，直至综合评价影像学和实验室检查数据以及受试者的临床状况(例如，肿瘤症状恶化)后研究者确定受试者缺乏临床获益，或直至受试者自愿退出研究治疗(以先发生者为准)。According to RECIST version 1.1, tumor efficacy evaluation is divided into CR (complete remission), PR (partial remission), SD (stable disease) and PD (progressive disease). For subjects with CR or PR, remission should be confirmed ≥4 weeks after the first recorded date of PR or CR. For subjects evaluated as PD, if the investigator and the sponsor believe that the subject may benefit from continued treatment, treatment may continue until the investigator determines that the subject lacks clinical benefit after a comprehensive evaluation of the imaging and laboratory test data and the subject's clinical condition (e.g., worsening of tumor symptoms), or until the subject voluntarily withdraws from the study treatment (whichever occurs first).
对于终止治疗的受试者，如果在终止日期前4周已进行影像学评估，则无需在治疗结束时进行影像学评估。无疾病进展证据而停止治疗的受试者将继续按计划接受后续影像学评估，直至开始新的抗肿瘤治疗、疾病进展、退出研究、死亡或研究结束，以先发生者为准。For subjects who discontinue treatment, if an imaging assessment has been performed 4 weeks before the discontinuation date, no imaging assessment is required at the end of treatment. Subjects who discontinue treatment without evidence of disease progression will continue to receive follow-up imaging assessments as planned until the start of new anti-tumor treatment, disease progression, withdrawal from the study, death, or end of the study, whichever occurs first.
4.8数据分析/统计方法 4.8 Data Analysis/Statistical Methods
本研究将采用描述性统计方法进行分析。将提供汇总统计量，分类变量报告受试者例数和百分比，连续变量报告受试者例数、平均值、标准差、中位数、最小值和最大值。使用Kaplan-Meier方法描述时间-事件变量。This study will use descriptive statistical methods for analysis. Summary statistics will be provided, and categorical variables will report the number of subjects and percentages, and continuous variables will report the number of subjects, mean, standard deviation, median, minimum and maximum values. The Kaplan-Meier method will be used to describe time-to-event variables.
安全性safety
使用国际监管活动医学词典(MedDRA)对不良事件进行编码。将研究期间发生的所有AE纳入列表中，并按MedDRA系统器官分类和首选术语汇总。将提供临床实验室检查、生命体征、ECOG体能状态评分、ECG等的额外安全性总结。Adverse events will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). All AEs occurring during the study will be included in a list and summarized by MedDRA system organ class and preferred term. Additional safety summaries of clinical laboratory tests, vital signs, ECOG performance status score, ECG, etc. will be provided.
疗效Efficacy
使用描述性统计方法分析疗效指标，包括ORR、DCR、PFS和OS。将在末例受试者接受首次输注后约16周或研究结束时(以先发生者为准)，对确认的最佳总缓解ORR进行主要疗效分析。采用Kaplan-Meier法总结DOR、PFS和OS。 Descriptive statistical methods were used to analyze efficacy indicators, including ORR, DCR, PFS, and OS. The primary efficacy analysis will be performed on the confirmed best overall response ORR approximately 16 weeks after the last subject received the first infusion or at the end of the study (whichever occurs first). The Kaplan-Meier method was used to summarize DOR, PFS, and OS.

Claims (12)

  1. 一种治疗恶性肿瘤的方法，其中，所述方法包括向有此需要的患者施用包含抗PVRIG/抗TIGIT的双特异性抗体的其药物组合物，所述药物组合物包含：A method for treating a malignant tumor, wherein the method comprises administering to a patient in need thereof a pharmaceutical composition comprising an anti-PVRIG/anti-TIGIT bispecific antibody, wherein the pharmaceutical composition comprises:
    (i)约10mg/ml至约200mg/ml的抗PVRIG/抗TIGIT的双特异性抗体；(i) about 10 mg/ml to about 200 mg/ml of an anti-PVRIG/anti-TIGIT bispecific antibody;
    (ii)缓冲体系为约10mM至约100mM的醋酸-醋酸钠缓冲液，pH5.0；(ii) the buffer system is about 10 mM to about 100 mM acetic acid-sodium acetate buffer, pH 5.0;
    (iii)约6％至约10％(w/v)蔗糖；和(iii) about 6% to about 10% (w/v) sucrose; and
    (iv)约0.01％至约0.10％(w/v)聚山梨酯80；(iv) about 0.01% to about 0.10% (w/v) polysorbate 80;
    所述抗PVRIG/抗TIGIT的双特异性抗体包含：The anti-PVRIG/anti-TIGIT bispecific antibody comprises:
    (a)第一抗原结合部分，其包括重链可变区(VH)和轻链可变区(VL)，所述VH和VL形成抗TIGIT抗原结合域；包含SEQ ID NO：9所示序列的HCDR1、SEQ ID NO：10所示序列的HCDR2、SEQ ID NO：11所示序列的HCDR3、SEQ ID NO：12所示序列的LCDR1、SEQ ID NO：13所示序列的LCDR2和SEQ ID NO：14所示序列的LCDR3：(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; comprising a HCDR1 of the sequence shown in SEQ ID NO: 9, a HCDR2 of the sequence shown in SEQ ID NO: 10, a HCDR3 of the sequence shown in SEQ ID NO: 11, a LCDR1 of the sequence shown in SEQ ID NO: 12, a LCDR2 of the sequence shown in SEQ ID NO: 13, and a LCDR3 of the sequence shown in SEQ ID NO: 14:
    (b)第二抗原结合部分，其包括特异性结合PVRIG的VHH；包含SEQ ID NO：6所示序列的CDR1、SEQ ID NO：7所示序列的CDR2和SEQ ID NO：8所示序列的CDR3；(b) a second antigen-binding portion, which includes a VHH that specifically binds to PVRIG; comprising a CDR1 of the sequence shown in SEQ ID NO: 6, a CDR2 of the sequence shown in SEQ ID NO: 7, and a CDR3 of the sequence shown in SEQ ID NO: 8;
    所述恶性肿瘤为转移性或局部晚期实体瘤，所述的实体瘤包括非小细胞肺癌(NSCLC)，肝细胞癌(HCC)，黑色素瘤，或人咽头癌。The malignant tumor is a metastatic or locally advanced solid tumor, and the solid tumor includes non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), melanoma, or human pharyngeal cancer.
  2. 根据权利要求1所述的方法，其中，所述抗PVRIG/抗TIGIT的双特异性抗体中，The method according to claim 1, wherein the anti-PVRIG/anti-TIGIT bispecific antibody
    (a)第一抗原结合部分为TIGIT抗体，包括两条VH和两条VL，所述VH包含SEQ ID NO：5所示氨基酸或与序列SEQ ID NO：5所示氨基酸相比至少90％同一性的氨基酸序列；所述VL包含SEQ ID NO：4所示氨基酸序列或与序列SEQ ID NO：4所示氨基酸至少90％同一性的氨基酸序列；(a) the first antigen-binding portion is a TIGIT antibody, comprising two VHs and two VLs, wherein the VHs comprise the amino acids shown in SEQ ID NO: 5 or an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO: 5; and the VLs comprise the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO: 4;
    (b)特异性结合PVRIG的VHH的C端连接于所述TIGIT抗体重链的N端，包含SEQ ID NO：3所示氨基酸序列或与序列SEQ ID NO：3所示氨基酸至少90％同一性的氨基酸序列。(b) The C-terminus of the VHH that specifically binds to PVRIG is connected to the N-terminus of the TIGIT antibody heavy chain, and comprises the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence that is at least 90% identical to the amino acid shown in SEQ ID NO: 3.
  3. 根据权利要求1或2所述的方法，其中，所述的药物组合物包含约50mg/mL的所述抗PVRIG/抗TIGIT的双特异性抗体、8％w/v蔗糖、和0.04％的聚山梨酯80；缓冲体系为20mM的醋酸-醋酸钠缓冲液，pH5.0；所述药物组合物的剂型为液体形式或冻干粉制剂。The method according to claim 1 or 2, wherein the pharmaceutical composition comprises about 50 mg/mL of the anti-PVRIG/anti-TIGIT bispecific antibody, 8% w/v sucrose, and 0.04% polysorbate 80; the buffer system is 20 mM acetic acid-sodium acetate buffer, pH 5.0; the dosage form of the pharmaceutical composition is a liquid form or a lyophilized powder preparation.
  4. 根据权利要求1-3任一所述的方法，其中，所述抗PVRIG/抗TIGIT的双特异性抗体的单次给药剂量为1mg-800mg，优选地，为5mg-800mg，10mg-800mg，20mg-800mg，30mg-800mg，5mg-700mg，10mg-700mg，20mg-700mg，30mg-700mg，5mg-600mg，10mg-600mg，20mg-600mg，30mg-600mg，50mg-600mg，100mg-600mg，300mg-600mg，10mg-300mg，30mg-300mg，100mg-300mg，10mg-100mg，30mg-100mg，或10mg-30mg。 The method according to any one of claims 1 to 3, wherein the single dosage of the anti-PVRIG/anti-TIGIT bispecific antibody is 1 mg-800 mg, preferably 5 mg-800 mg, 10 mg-800 mg, 20 mg-800 mg, 30 mg-800 mg, 5 mg-700 mg, 10 mg-700 mg, 20 mg-700 mg, 30 mg-700 mg, 5 mg-600 mg, 10 mg-600 mg, 20 mg-600 mg, 30 mg-600 mg, 50 mg-600 mg, 100 mg-600 mg, 300 mg-600 mg, 10 mg-300 mg, 30 mg-300 mg, 100 mg-300 mg, 10 mg-100 mg, 30 mg-100 mg, or 10 mg-30 mg.
  5. 根据权利要求1或2所述的方法，其中，所述抗PVRIG/抗TIGIT的双特异性抗体的单次给药剂量为约1mg，5mg，10mg，15mg，20mg，30mg，40mg，50mg，60mg，70mg，80mg，90mg，100mg，150mg，200mg，250mg，300mg，350mg，400mg，450mg，500mg，550mg，600mg，650mg，700mg，750mg，或800mg。The method according to claim 1 or 2, wherein the single administration dose of the anti-PVRIG/anti-TIGIT bispecific antibody is about 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, or 800 mg.
  6. 根据权利要求1所述的方法，其中，所述非小细胞肺癌(NSCLC)为：The method according to claim 1, wherein the non-small cell lung cancer (NSCLC) is:
    经组织学或细胞学证实的不可治愈的晚期NSCLC；或Histologically or cytologically confirmed incurable advanced NSCLC; or
    接受至少1种但不超过2种系统抗肿瘤治疗后疾病进展的NSCLC，且无驱动基因突变；或接受至少1种已确定的标准抗肿瘤靶向治疗失败，或不适合目前的标准治疗的NSCLC，且有驱动基因突变。NSCLC with disease progression after at least 1 but no more than 2 systemic anti-tumor therapies and without driver gene mutations; or NSCLC with driver gene mutations after failure of at least 1 established standard anti-tumor targeted therapy or not suitable for current standard treatment.
  7. 根据权利要求1所述的方法，其中，所述肝细胞癌(HCC)为：The method according to claim 1, wherein the hepatocellular carcinoma (HCC) is:
    为经组织学或细胞学证实的HCC；HCC confirmed by histology or cytology;
    为经过至少1种含抗血管生成疗法的治疗失败，但不超过2种全身抗肿瘤治疗的HCC；HCC that has failed at least 1 anti-angiogenic therapy but no more than 2 systemic anti-tumor therapies;
    基于巴塞罗那临床肝癌(BCLC)分期系统的B期或C期，不适合手术或局部治疗，或在手术和/或局部治疗后进展的HCC；HCC stage B or C based on the Barcelona Clinic Liver Cancer (BCLC) staging system, not suitable for surgery or local therapy, or progressing after surgery and/or local therapy;
    Child-Pugh评分≤7且无肝性脑病的HCC。HCC with Child-Pugh score ≤7 and without hepatic encephalopathy.
  8. 根据权利要求1-7任一项所述的方法，其中，所述药物组合物的剂型规格(全物质含量)为100-400mg/瓶，优选100-300mg/瓶，150-350mg/瓶，200-350mg/瓶，200-300mg/瓶或250-300mg/瓶。The method according to any one of claims 1 to 7, wherein the dosage form specification (total substance content) of the pharmaceutical composition is 100-400 mg/bottle, preferably 100-300 mg/bottle, 150-350 mg/bottle, 200-350 mg/bottle, 200-300 mg/bottle or 250-300 mg/bottle.
  9. 根据权利要求8所述的方法，其中，所述其药物组合物的剂型规格(全物质含量)为约100mg/瓶、150mg/瓶、180mg/瓶、200mg/瓶、250mg/瓶、300mg/瓶、350mg/瓶或400mg/瓶；优选地，所述药物组合物为单剂量剂型，每剂包含能够给予患者单次施用量的抗体量。The method according to claim 8, wherein the dosage form specification (full substance content) of the pharmaceutical composition is about 100 mg/bottle, 150 mg/bottle, 180 mg/bottle, 200 mg/bottle, 250 mg/bottle, 300 mg/bottle, 350 mg/bottle or 400 mg/bottle; preferably, the pharmaceutical composition is a single-dose dosage form, each dose containing an amount of antibody that can be given to a patient in a single administration.
  10. 权利要求1-9任一项所述的方法，其中，所述抗PVRIG/抗TIGIT抗体或其药物组合物的给药频率为约每一周一次、每两周一次、每三周一次、每四周一次或一个月一次，优选为每周给药一次。The method according to any one of claims 1 to 9, wherein the frequency of administration of the anti-PVRIG / anti-TIGIT antibody or its pharmaceutical composition is approximately once a week, once every two weeks, once every three weeks, once every four weeks or once a month, preferably once a week.
  11. 根据权利要求1‐10任一项所述的方法，其中，所述抗PVRIG/抗TIGIT抗体或其药物组合物通过静脉滴注、静脉注射、口服、皮下注射、肌内或瘤内注射给药，优选地，通过静脉输注给药。The method according to any one of claims 1 to 10, wherein the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition is administered by intravenous drip, intravenous injection, oral administration, subcutaneous injection, intramuscular or intratumoral injection, preferably, by intravenous infusion.
  12. 根据权利要求1-11所述的方法，其中，所述抗PVRIG/抗TIGIT抗体或其药物组合物 的给药周期为一周、二周、三周、四周、两个月、三个月、四个月、五个月、半年或更长时间，任选地，每个给药周期的时间相同或不同，且每个给药周期之间的间隔相同或不同。 The method according to claims 1-11, wherein the anti-PVRIG/anti-TIGIT antibody or its pharmaceutical composition The administration cycle is one week, two weeks, three weeks, four weeks, two months, three months, four months, five months, six months or longer, optionally, the time of each administration cycle is the same or different, and the interval between each administration cycle is the same or different.
PCT/CN2024/073012 2023-01-19 2024-01-18 Use of anti-pvrig/anti-tigit bispecific antibody in treatment of malignant tumors WO2024153182A1 (en)

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