WO2022258049A1

WO2022258049A1 - Genetically modified non-human animal with human or chimeric pvrig

Info

Publication number: WO2022258049A1
Application number: PCT/CN2022/098158
Authority: WO
Inventors: Yifu Zhang; Jiawei Yao; Chonghui LIU
Original assignee: Biocytogen Pharmaceuticals (Beijing) Co., Ltd.
Priority date: 2021-06-10
Filing date: 2022-06-10
Publication date: 2022-12-15
Also published as: CN115010800A

Abstract

Provided are genetically modified non-human animals that express a human or chimeric (e.g., humanized) PVRIG, and methods of use thereof.

Description

GENETICALLY MODIFIED NON-HUMAN ANIMAL WITH HUMAN OR CHIMERIC PVRIG

CLAIM OF PRIORITY

This application claims the benefit of Chinese Patent Application App. No. 202110650639.4, filed on June 10, 2021 and Chinese Patent Application App. No. 202111294418.4, filed on November 3, 2021. The entire contents of the foregoing applications are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animal expressing human or chimeric (e.g., humanized) poliovirus receptor-related immunoglobulin domain-containing (PVRIG) , and methods of use thereof.

BACKGROUND

The immune system has developed multiple mechanisms to prevent deleterious activation of immune cells. One such mechanism is the intricate balance between positive and negative co-stimulatory signals delivered to immune cells. Targeting the stimulatory or inhibitory pathways for the immune system is considered to be a potential approach for the treatment of various diseases, e.g., cancers and autoimmune diseases.

The traditional drug research and development for these stimulatory or inhibitory receptors typically use in vitro screening approaches. However, these screening approaches cannot provide the body environment (such as tumor microenvironment, stromal cells, extracellular matrix components and immune cell interaction, etc. ) , resulting in a higher rate of failure in drug development. In addition, in view of the differences between humans and animals, the test results obtained from the use of conventional experimental animals for in vivo pharmacological test may not reflect the real disease state and the interaction at the targeting sites, resulting in that the results in many clinical trials are significantly different from the animal experimental results. Therefore, the development of humanized animal models that are suitable for human antibody screening and evaluation will significantly improve the efficiency of new drug development and reduce the cost for drug research and development.

SUMMARY

This disclosure is related to an animal model with human PVRIG or chimeric PVRIG. The animal model can express human PVRIG or chimeric PVRIG (e.g., humanized PVRIG) protein in its body. It can be used in the studies on the function of PVRIG gene, and can be used in the screening and evaluation of anti-human PVRIG antibodies. In addition, the animal models prepared by the methods described herein can be used in drug screening, pharmacodynamics studies, treatments for immune-related diseases (e.g., autoimmune diseases) , and cancer therapy for human PVRIG target sites; they can also be used to facilitate the development and design of new drugs, and save time and cost. In summary, this disclosure provides a powerful tool for studying the function of PVRIG protein and a platform for screening cancer drugs.

In one aspect, the disclosure is related to a genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric PVRIG.

In some embodiments, the sequence encoding the human or chimeric PVRIG is operably linked to an endogenous regulatory element at the endogenous PVRIG gene locus in the at least one chromosome.

In some embodiments, the sequence encoding a human or chimeric PVRIG comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human PVRIG (NP_076975.2 (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding a human or chimeric PVRIG comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.

In some embodiments, the sequence encoding a human or chimeric PVRIG comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 1-169 of SEQ ID NO: 2.

In some embodiments, the animal is a mammal, e.g., a monkey, a rodent, or a mouse.

In some embodiments, the animal is a mouse.

In some embodiments, the animal does not express endogenous PVRIG.

In some embodiments, the animal has one or more cells expressing human or chimeric PVRIG.

In some embodiments, the animal has one or more cells expressing human or chimeric PVRIG, and a human PVRIG ligand can bind to the expressed human or chimeric PVRIG.

In some embodiments, the animal has one or more cells expressing human or chimeric PVRIG, and an endogenous PVRIG ligand can bind to the expressed human or chimeric PVRIG.

In one aspect, the disclosure is related to a genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous PVRIG with a sequence encoding a corresponding region of human PVRIG at an endogenous PVRIG gene locus.

In some embodiments, the sequence encoding the corresponding region of human PVRIG is operably linked to an endogenous regulatory element at the endogenous PVRIG locus, and one or more cells of the animal expresses a chimeric PVRIG.

In some embodiments, the animal does not express endogenous PVRIG.

In some embodiments, the replaced sequence encodes all or a portion of the extracellular region (with or without signal peptide) of endogenous PVRIG.

In some embodiments, the animal has one or more cells expressing a chimeric PVRIG having a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region.

In some embodiments, the chimeric PVRIG has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 contiguous amino acids that are identical to a contiguous sequence present in human PVRIG.

In some embodiments, the sequence encoding a region of endogenous PVRIG comprises exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous PVRIG gene.

In some embodiments, the animal is a mouse, and the sequence encoding a region of endogenous PVRIG starts within exon 1 and ends within exon 3 of the endogenous mouse PVRIG gene.

In some embodiments, the animal is heterozygous with respect to the replacement at the endogenous PVRIG gene locus.

In some embodiments, the animal is homozygous with respect to the replacement at the endogenous PVRIG gene locus.

In one aspect, the disclosure is related to a method for making a genetically-modified, non-human animal, comprising: replacing in at least one cell of the animal, at an endogenous PVRIG gene locus, a sequence encoding a region of an endogenous PVRIG with a sequence encoding a corresponding region of human PVRIG.

In some embodiments, the sequence encoding the corresponding region of human PVRIG comprises exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6, or a part thereof, of a human PVRIG gene.

In some embodiments, the sequence encoding the corresponding region of human PVRIG starts within exon 2 and ends within exon 4 of a human PVRIG gene.

In some embodiments, the sequence encoding the corresponding region of human PVRIG encodes amino acids 1-169 of SEQ ID NO: 2.

In some embodiments, the replaced region of an endogenous PVRIG is located within the extracellular region.

In some embodiments, the sequence encoding a region of endogenous PVRIG comprises exon 1, exon 2, exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous PVRIG gene.

In some embodiments, the animal is a mouse, and the replaced sequence of an endogenous PVRIG starts within exon 1 and ends within exon 3 of the endogenous mouse PVRIG gene.

In one aspect, the disclosure is related to a non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric PVRIG polypeptide, wherein the chimeric PVRIG polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human PVRIG, wherein the animal expresses the chimeric PVRIG.

In some embodiments, the chimeric PVRIG polypeptide has at least 50, at least 100, or at least 150 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human PVRIG.

In some embodiments, the chimeric PVRIG polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-169 of SEQ ID NO: 2.

In some embodiments, the nucleotide sequence is operably linked to an endogenous PVRIG regulatory element of the animal.

In some embodiments, the chimeric PVRIG polypeptide comprises an endogenous PVRIG transmembrane region and/or an endogenous PVRIG cytoplasmic region.

In some embodiments, the nucleotide sequence is integrated to an endogenous PVRIG gene locus of the animal.

In some embodiments, the chimeric PVRIG has at least one mouse PVRIG activity and/or at least one human PVRIG activity.

In one aspect, the disclosure is related to a method of making a genetically-modified non-human animal cell that expresses a chimeric PVRIG, the method comprising: replacing at an endogenous PVRIG gene locus, a nucleotide sequence encoding a region of endogenous PVRIG with a nucleotide sequence encoding a corresponding region of human PVRIG, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric PVRIG, wherein the non-human animal cell expresses the chimeric PVRIG.

In some embodiments, the chimeric PVRIG comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region.

In some embodiments, the nucleotide sequence encoding the chimeric PVRIG is operably linked to an endogenous PVRIG regulatory region, e.g., promoter.

In some embodiments, the animal further comprises a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein is PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPα, CTLA4, or CD226.

In some embodiments, the additional human or chimeric protein is PD-1 and/or TIGIT, and the animal expresses the human or chimeric PD-1 and/or TIGIT.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-PVRIG antibody for the treatment of cancer, comprising: administering the anti-PVRIG antibody to the animal described herein, wherein the animal has a cancer; and determining the inhibitory effects of the anti-PVRIG antibody to the cancer.

In some embodiments, one or more cells (e.g., T or NK cells) in the cancer express PVRIG.

In some embodiments, the cancer comprises one or more cancer cells that are injected into the animal.

In some embodiments, determining the inhibitory effects of the anti-PVRIG antibody to the cancer involves measuring the tumor volume in the animal.

In some embodiments, the cancer is head and neck cancer, thyroid cancer, liver cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, glioma, ovarian cancer, stomach cancer, carcinoid, urothelial cancer, breast cancer, endometrial cancer, testis cancer, pancreatic cancer, cervical cancer, renal cancer, lymphoma or skin cancer.

In one aspect, the disclosure is related to a method of determining effectiveness of an anti-PVRIG antibody and an additional therapeutic agent for the treatment of cancer, comprising administering the anti-PVRIG antibody and the additional therapeutic agent to the animal described herein, wherein the animal has a cancer; and determining the inhibitory effects on the cancer.

In some embodiments, the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) .

In some embodiments, the animal further comprises a sequence encoding a human or chimeric TIGIT.

In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody or an anti-TIGIT antibody.

In some embodiments, the cancer comprises one or more cancer cells that express a PVRIG ligand (e.g., PVRL2) .

In some embodiments, the cancer is caused by injection of one or more cancer cells into the animal.

In some embodiments, determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal.

In some embodiments, the animal has head and neck cancer, thyroid cancer, liver cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, glioma, ovarian cancer, stomach cancer, carcinoid, urothelial cancer, breast cancer, endometrial cancer, testis cancer, pancreatic cancer, cervical cancer, renal cancer, lymphoma or skin cancer.

In one aspect, the disclosure is related to a protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

an amino acid sequence set forth in SEQ ID NO: 2 or 11;

an amino acid sequence that is at least 90%identical to SEQ ID NO: 2 or 11;

an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or 11;

an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

an amino acid sequence that comprises a substitution, a deletion and /or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 2 or 11.

In one aspect, the disclosure is related to a nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

a sequence that encodes the protein described herein;

SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10;

a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10; and

a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10.

In one aspect, the disclosure is related to a cell comprising the protein described herein and/or the nucleic acid described herein.

In one aspect, the disclosure is related to an animal comprising the protein described herein and/or the nucleic acid described herein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing mouse PVRIG gene locus and human PVRIG gene locus.

FIG. 2 is a schematic diagram showing humanized PVRIG gene locus.

FIG. 3 is a schematic diagram showing a PVRIG gene targeting strategy.

FIG. 4 shows Southern Blot results of cells after recombination using the 5' Probe, 3' Probe, Neo Probe. 1-B08, 3-E06, 3-F11 and 3-G06 are clone numbers. WT is a wild-type control.

FIG. 5 is a schematic diagram showing the FRT recombination process.

FIG. 6A shows PCR identification results of F1 generation mice by primers WT-F and WT-R. F1-01, F1-02, and F1-03 are mouse numbers. M is a marker. PC is a positive control (Neo cassette removed) . WT is a wild-type control. H ₂O is a water control.

FIG. 6B shows PCR identification results of F1 generation mice by primers WT-F and Mut-R. F1-01, F1-02, and F1-03 are mouse numbers. M is a marker. PC is a positive control (Neo cassette removed) . WT is a wild-type control. H ₂O is a water control.

FIG. 6C shows PCR identification results of F1 generation mice by primers Frt-F and Frt-R. F1-01, F1-02, and F1-03 are mouse numbers. M is a marker. PC is a positive control (Neo cassette removed) . WT is a wild-type control. H ₂O is a water control.

FIG. 6D shows PCR identification results of F1 generation mice by primers Flp-F and Flp-R. F1-01, F1-02, and F1-03 are mouse numbers. M is a marker. PC is a positive control (Neo cassette removed) . WT is a wild-type control. H ₂O is a water control.

FIGS. 7A-7C show RT-PCR identification results of wild-type C57BL/6 mice (+/+) or PVRIG gene humanized homozygous mice (H/H) to detect expression of mouse PVRIG (PCR-F2/PCR-R2) , humanized PVRIG (PCR-F1/PCR-R1) , and Mouse GAPDH (GAPDH-F/GAPDH-R) . H ₂O is a water control.

FIG. 8 shows the percentages of leukocyte subtypes in the spleen of C57BL/6 wild-type mice and PVRIG gene humanized homozygous mice (B-hPVRIG) , as determined by flow cytometry.

FIG. 9 shows the percentages of T cell subtypes in the spleen of C57BL/6 wild-type mice and PVRIG gene humanized homozygous mice (B-hPVRIG) , as determined by flow cytometry.

FIG. 10 shows the percentages of leukocyte subtypes in the peripheral blood of C57BL/6 wild-type mice and PVRIG gene humanized homozygous mice (B-hPVRIG) , as determined by flow cytometry.

FIG. 11 shows the percentages of T cell subtypes in the peripheral blood of C57BL/6 wild-type mice and PVRIG gene humanized homozygous mice (B-hPVRIG) , as determined by flow cytometry.

FIG. 12 shows the percentages of leukocyte subtypes in the lymph nodes of C57BL/6 wild-type mice and PVRIG gene humanized homozygous mice (B-hPVRIG) , as determined by flow cytometry.

FIG. 13 shows the percentages of T cell subtypes in the lymph nodes of C57BL/6 wild-type mice and PVRIG gene humanized homozygous mice (B-hPVRIG) , as determined by flow cytometry.

FIG. 14 shows the average body weight of PVRIG gene humanized homozygous mice that were xenografted with mouse colon cancer cells MC38, and then treated with COM-701 at 30 mg/kg (G2) or SRF-813 at 30 mg/kg (G3) . 30 mg/kg human IgG (hIgG) was administered as a control (G1) .

FIG. 15 shows the body weight change of PVRIG gene humanized homozygous mice that were xenografted with mouse colon cancer cells MC38, and then treated with COM-701 at 30 mg/kg (G2) or SRF-813 at 30 mg/kg (G3) . 30 mg/kg human IgG (hIgG) was administered as a control (G1) .

FIG. 16 shows the tumor volume of PVRIG gene humanized homozygous mice that were xenografted with mouse colon cancer cells MC38, and then treated with COM-701 at 30 mg/kg (G2) or SRF-813 at 30 mg/kg (G3) . 30 mg/kg human IgG (hIgG) was administered as a control (G1) .

FIG. 17 shows the alignment between human PVRIG amino acid sequence (NP_076975.2; SEQ ID NO: 2) and mouse PVRIG amino acid sequence (NP_001365367.1; SEQ ID NO: 1) .

FIG. 18 shows the alignment between human PVRIG amino acid sequence (NP_076975.2; SEQ ID NO: 2) and rat PVRIG amino acid sequence (XP_006249093.1; SEQ ID NO: 31) .

DETAILED DESCRIPTION

Tumor cells evade immune surveillance. Cancer immunotherapies including immune-checkpoint blockade have been successful in the clinic, underscoring the value of the immune system in surveillance and elimination of cancer. Monoclonal antibodies blocking immune-checkpoint pathways have been or are being developed that rescue dormant antitumor T-cell effector responses.

Among the immune-checkpoint pathways, a group of receptors and ligands within the nectin and nectin-like family are under intense investigation. Receptors within this family include DNAM-1 (CD226) , CD96 (TACTILE) , TIGIT, and PVRIG (CD112R) . Of these molecules, DNAM is a costimulatory receptor that binds to two ligands, PVR (CD155) and PVRL2 (CD112) . In contrast to DNAM-1, two inhibitory receptors in this family, TIGIT and PVRIG, have been shown to dampen human lymphocyte function. TIGIT is reported to have a high-affinity interaction with PVR, a weaker affinity for PVRL2 and PVRL3, and inhibits both T-cell and NK cell responses through signaling of its intracellular tail or by inhibition of PVR-DNAM interactions to prevent DNAM signaling. PVRIG binds only to PVRL2 with high affinity and suppresses T-cell function. The affinities of TIGIT for PVR and PVRIG for PVRL2, respectively, are higher than the affinity of DNAM to either of its ligands. Collectively, these data indicate that there are three mechanisms by which TIGIT or PVRIG can suppress T-cell function: (i) direct inhibitory signaling through inhibitory motifs contained within their intracellular domains; (ii) sequestration of ligand binding from DNAM-1; and (iii) disruption of DNAM homodimerization and signaling. Within this family, PVR is also a ligand for CD96, whose immunomodulatory role on lymphocytes is less clear.

PVRIG has a different expression profile on murine T-cell subsets compared with TIGIT and that its dominant ligand, PVRL2, is upregulated on myeloid and tumor cells in the tumor microenvironment (TME) . Furthermore, inhibition of PVRIG-PVRL2 interaction reduced tumor growth in a

T cell-dependent manner or with synergistic effects when combined with PD-L1 blockade. Collectively, these data show that mouse PVRIG is an inhibitory receptor that regulates T-cell antitumor responses.

Experimental animal models are an indispensable research tool for studying the effects of these antibodies (e.g., PVRIG antibodies) . Common experimental animals include mice, rats, guinea pigs, hamsters, rabbits, dogs, monkeys, pigs, fish and so on. However, there are many differences between human and animal genes and protein sequences, and many human proteins cannot bind to the animal's homologous proteins to produce biological activity, leading to that the results of many clinical trials do not match the results obtained from animal experiments. A large number of clinical studies are in urgent need of better animal models. With the continuous development and maturation of genetic engineering technologies, the use of human cells or genes to replace or substitute an animal's endogenous similar cells or genes to establish a biological system or disease model closer to human, and establish the humanized experimental animal models (humanized animal model) has provided an important tool for new clinical approaches or means. In this context, the genetically engineered animal model, that is, the use of genetic manipulation techniques, the use of human normal or mutant genes to replace animal homologous genes, can be used to establish the genetically modified animal models that are closer to human gene systems. The humanized animal models have various important applications. For example, due to the presence of human or humanized genes, the animals can express or express in part of the proteins with human functions, so as to greatly reduce the differences in clinical trials between humans and animals, and provide the possibility of drug screening at animal levels.

This disclosure relates to transgenic non-human animal with human or chimeric (e.g., humanized) PVRIG, and methods of use thereof.

PVRIG

Poliovirus receptor-related immunoglobulin domain-containing (PVRIG) , also known as CD112R, has recently been identified as an immune checkpoint molecule with potential for therapeutic development. In humans, PVRIG is expressed on T cells (predominantly CD8+ T cells) and natural killer (NK) cells, but not on B cells, monocytes or neutrophils. PVRIG binds to a single ligand, poliovirus receptor-related 2 (PVRL2, also known as CD112 or Nectin-2) , and exerts an inhibitory effect on cytotoxic lymphocyte activity, likely via an ITIM-like motif in its intracellular domain. PVRL2 is an adhesion molecule involved in the formation of cell-cell junctions, and is overexpressed in various cancers. PVRL2 is also a ligand of the co-activating receptor DNAX accessory molecule 1 (DNAM-1) and weakly binds another inhibitory receptor, T-cell immunoreceptor with Ig and ITIM domains (TIGIT) . The inhibitory effect of PVRL2 was predominantly mediated by PVRIG and not TIGIT. DNAM-1 and TIGIT (but not PVRIG) also bind to a closely related molecule, poliovirus receptor (PVR, also known as CD155 or Necl-5) . Competition studies have determined that PVR has higher affinity for TIGIT than DNAM-1, and PVRL2 has a higher affinity for PVRIG than DNAM-1, suggesting that the inhibitory signal is dominant.

PVRIG was identified as immunoreceptors that are involved in regulating lymphocyte function in the context of cancer. Human and cynomolgus PVRIG share high sequence identity, and both proteins have a conserved ITIM in the intracellular signaling domain, which is lacking in the mouse homolog. PVRIG is a 36 kD transmembrane monomer composed of a single extracellular IgV domain, one transmembrane domain and a long intracellular domain. The human PVRIG intracellular domain comprises two tyrosine residues, one of which is part of an ITIM-like motif and a potential site for phosphatases. The extracellular domain sequences of human and mouse PVRIG exhibit ～65.3%homology. In addition, phylogenetic tree analysis of the first IgV of the PVR family revealed that PVRIG is closely related to PVR-like proteins.

Mouse PVRIG has a 59%protein identity with human PVRIG and a truncated intracellular signaling domain that contains phosphorylated tyrosine but lacks an ITIM, suggesting that mouse PVRIG may have a reduced role as a DNAM1 pathway checkpoint receptor compared with human PVRIG. At steady state, expression of murine PVRIG is detected on both T and NK cells, whereas in peripheral immune tissues, NK but not T cells express PVRIG. Upon activation, CD8+ T cells upregulate PVRIG expression, although at a much slower rate compared with related coinhibitory checkpoints. The relative expression of mouse PVRIG on TILs is lower than that seen in humans, and mouse PVRL2 in the TME and mouse tumor cell lines is lower than the expression detected in human TME, further supporting the idea that PVRIG plays a diminished role in mice compared with its role in humans. These findings, including the acquisition of an ITIM in human PVRIG and higher levels of human PVRIG and PVRL2 in the TME, suggest that greater effects of PVRIG inhibition may be seen in a human tumor setting relative to mouse preclinical models.

In the human setting, as in the mouse setting, results indicate that inhibition of this pathway may result in enhanced antitumor immunity. The activating effect of PVRIG blockade on CD8+ T cells has been demonstrated in vitro with antibodies that block the PVRIG/PVRL2 interaction. The ability of anti-PVRIG mAbs to promote T-cell responses, either individually or in combination with other immune checkpoints, was assessed using multiple human T cell-based assays with both TILs and T cells from healthy donors. In all assay systems, PVRIG blockade increased T-cell proliferation, cytokine secretion, and cytotoxicity. Although clinical monotherapy effects are rarely observed with inhibitory receptor antagonists other than anti-PD-1, numerous pathways modulate immune responses, suggesting that combinatorial approaches may increase rates of response. Compared with blockade of PVRIG, TIGIT, or PD-1 alone, the dual and triple combination of anti-PVRIG with anti-PD-1 or with anti-TIGIT further increased cytokine production and T cell-mediated killing of PVRL2+PVR+ tumor target cells.

In addition to PVRIG's role in regulating T-cell responses, PVRIG blockade significantly enhances NK cell-mediated killing of PVRL2+ cancer cells. Also, blockade of PVRIG and TIGIT alone or in combination enhances trastuzumab-triggered antitumor response by human NK cells. Thus, PVRIG and TIGIT receptors regulate NK-cell functions and that NK activation may be a determinant of clinical efficacy for inhibitors targeting each receptor.

Hormonally regulated tumors, such as ovarian, endometrial, and breast cancers, along with kidney and lung tumors, demonstrated the highest PVRIG expression on T and NK cells. PVRIG expression in tumors was significantly increased on TILs compared with T cells infiltrating normal adjacent tissue, further highlighting its potential as a checkpoint receptor on lymphocytes. Moreover, PVRIG was coexpressed with PD-1 and TIGIT on TILs, peripheral memory T cells, and activated T cells. These data indicate that all three inhibitory molecules play a role in regulating the immune response and provide a rationale for the dual and triple blockade of these checkpoint receptors, depending on the ligand expression pattern in the TME. PVRL2 is frequently expressed in various malignancies, and specifically PVRL2/PVR mRNA ratios are generally highest in hormonally regulated cancers, which was further validated by both IHC and flow cytometry. This, together with PVRIG levels in those tumors, suggests that these cancers are promising indications for PVRIG-blocking antibodies. In addition, increased RNA and protein PVRL2 expression levels were demonstrated in cancer relative to normal tissues, with expression seen in both PD-L1+ and PD-L1-patient samples across tumor types. These analyses highlighted cancers in which PVRL2 might primarily regulate the immune response, and how this pathway may be relevant in patients who are PD-L1-or those who develop PD-1 resistance.

Based on the supportive preclinical data, an anti-PVRIG antibody was developed for clinical testing. COM701 is a humanized anti-PVRIG hinge stabilized IgG4 mAb that binds specifically to human and cynomolgus monkey PVRIG and disrupts the binding of PVRIG to PVRL2. Because PVRIG is predominantly expressed on CD8+ T and NK cells, and its expression is relatively low on Tregs, an IgG4 backbone was selected to avoid potential depletion of effector cells. COM701 inhibits the binding of PVRL2 to PVRIG in a dose-dependent manner with complete inhibition of PVRIG/PVRL2 interaction observed at saturating levels of COM701. It is currently in phase I clinical testing, both as a monotherapy and in combination with the anti-PD-1 drug nivolumab (NCT03667716) . Thus, PVRIG antibodies can be potentially useful as cancer therapies.

A detailed description of PVRIG and its function can be found, e.g., in Alteber, Zoya, et al. "Therapeutic targeting of checkpoint receptors within the DNAM1 axis. "Cancer discovery 11.5 (2021) : 1040-1051; and Murter, Benjamin, et al. "Mouse PVRIG has CD8+ T cell-specific coinhibitory functions and dampens antitumor immunity. " Cancer immunology research 7.2 (2019) : 244-256; each of which is incorporated by reference in its entirety.

In mice, PVRIG gene locus has five exons, exon 1, exon 2, exon 3, exon 4, and exon 5 (FIG. 1) . The mouse PVRIG protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of PVRIG. The amino acid sequence for mouse PVRIG is NP_001365367.1 (SEQ ID NO: 1) . The location for each exon and each region in the mouse PVRIG nucleotide sequence and amino acid sequence is listed below:

Table 1

The mouse PVRIG gene (Gene ID: 102640920) is located in Chromosome 5 of the mouse genome, which is located from 138340193 to 138346953 (GRCm39 (GCF_000001635.27) ) . The 5'-UTR is from 138, 340, 223 to 138, 340, 230, exon 1 is from 138, 340, 223 to 138, 340, 330, the first intron is from 138, 340, 331 to 138, 340, 417, exon 2 is from 138, 340, 418 to 138, 340, 771, the second intron is from 138, 340, 772 to 138, 340, 847, exon 3 is from 138, 340, 848 to 138, 340, 971, the third intron is from 138, 340, 972 to 138, 341, 051, exon 4 is from 138, 341, 052 to 138, 341, 109, the fourth intron is from 138, 341, 110 to 138, 341, 383, exon 5 is from 138, 341, 384 to 138, 341, 819, he 3’-UTR is from 138341453 to 138341819, based on transcript NM_001378438.1. All relevant information for mouse PVRIG locus can be found in the NCBI website with Gene ID: 102640920, which is incorporated by reference herein in its entirety.

The human PVRIG gene (Gene ID: 79037) is located in Chromosome 14 of the human genome, which is located from 100218625 to 100221490 of NC_000007.14 (GRCh38. p13 (GCF_000001405.39) ) . The 5'-UTR is from 100, 219, 236 to 100, 219, 400, and 100, 219, 713 to 100219910, exon 1 is from 100, 219, 236 to 100, 219, 400, the first intron is from 100, 219, 401 to 100, 219, 712, exon 2 is from 100, 219, 713 to 100, 220, 028, the second intron is from 100, 220, 029 to 100, 220, 113, exon 3 is from 100, 220, 114 to 100, 220, 464 , the third intron is from 100, 220, 465 to 100, 220, 546, exon 4 is from 100, 220, 547 to 100, 220, 673, the fourth intron is from 100, 220, 674 to 100, 220, 759, exon 5 is from 100, 220, 760 to 100, 220, 820, the fifth intron is from 100, 220, 821 to 100, 220, 927, exon 6 is from 100, 220, 928 to 100, 221, 490, the 3'-UTR is from 100221252 to 100221490, based on transcript NM_024070.3. All relevant information for human PVRIG locus can be found in the NCBI website with Gene ID: 79037, which is incorporated by reference herein in its entirety.

In human genomes, PVRIG gene (Gene ID: 79037) locus has six exons, exon 1, exon 2, exon 3, exon 4, exon 5, and exon 6 (FIG. 1) . The PVRIG protein also has an extracellular region, a transmembrane region, and a cytoplasmic region, and the signal peptide is located at the extracellular region of PVRIG. The amino acid sequence for human PVRIG is NP_076975.2 (SEQ ID NO: 2) . The location for each exon and each region in human PVRIG nucleotide sequence and amino acid sequence is listed below:

Table 2

FIG. 17 shows the alignment between human PVRIG amino acid sequence (NP_076975.2; SEQ ID NO: 2) and mouse PVRIG amino acid sequence (NP_001365367.1; SEQ ID NO: 1) . Thus, the corresponding amino acid residue or region between human and mouse PVRIG can be found in FIG. 17.

FIG. 18 shows the alignment between human PVRIG amino acid sequence (NP_076975.2; SEQ ID NO: 2) and rat PVRIG amino acid sequence (XP_006249093.1; SEQ ID NO: 31) . Thus, the corresponding amino acid residue or region between human and mouse PVRIG can be found in FIG. 18.

PVRIG genes, proteins, and locus of the other species are also known in the art. For example, the gene ID for PVRIG in Rattus norvegicus (rat) is 680923, the gene ID for PVRIG in Macaca mulatta (Rhesus monkey) is 711659, the gene ID for PVRIG in Canis lupus familiaris (dog) is 608526, and the gene ID for PVRIG in Equus caballus (horse) is 102149166. The relevant information for these genes (e.g., intron sequences, exon sequences, amino acid residues of these proteins) can be found, e.g., in NCBI database, which is incorporated by reference herein in its entirety.

The present disclosure provides human or chimeric (e.g., humanized) PVRIG nucleotide sequence and/or amino acid sequences. In some embodiments, the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence. In some embodiments, a “region” or “portion” of mouse exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, and/or cytoplasmic region are replaced by the corresponding human sequence. The term “region” or “portion” can refer to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or 5481 nucleotides, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, or 234 amino acid residues. In some embodiments, the “region” or “portion” can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical to exon 1, exon 2, exon 3, exon 4, exon 5, signal peptide, extracellular region, transmembrane region, or cytoplasmic region of mouse PVRIG gene; or exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, signal peptide, extracellular region, transmembrane region, or cytoplasmic region of human PVRIG gene. In some embodiments, a region, a portion, or the entire sequence of mouse exon 1, exon 2, exon 3, exon 4, and/or exon 5 (e.g., a portion of exon 1, exon 2 and a portion of exon 3) are replaced by human exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6, (e.g., a portion of exon 2, exon 3, and a portion of exon 4) sequence.

In some embodiments, the genetically-modified non-human animal described herein comprises a sequence encoding a humanized PVRIG protein. In some embodiments, the humanized PVRIG protein comprises a humanized extracellular region. In some embodiments, the humanized PVRIG protein comprises an endogenous signal peptide. In some embodiments, the humanized PVRIG protein comprises an endogenous transmembrane region. In some embodiments, the humanized PVRIG protein comprises an endogenous cytoplasmic region.

In some embodiments, the genetically-modified non-human animal described herein comprises a humanized PVRIG gene. In some embodiments, the humanized PVRIG gene comprises 5 exons. In some embodiments, the humanized PVRIG gene comprises humanized exon 1, humanized exon 2, humanized exon 3, humanized exon 4, and/or humanized exon 5.

In some embodiments, the present disclosure also provides a chimeric (e.g., humanized) PVRIG nucleotide sequence and/or amino acid sequences, wherein in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from mouse PVRIG mRNA sequence, mouse PVRIG amino acid sequence (e.g., SEQ ID NO: 1) , or a portion thereof (e.g., a portion of exon 3, exon 4 and exon 5 of mouse PVRIG gene) ; and in some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%of the sequence are identical to or derived from human PVRIG mRNA sequence, human PVRIG amino acid sequence (e.g., SEQ ID NO: 2) , or a portion thereof (e.g., a portion of exon 2, exon 3, and a portion of exon 4 of human PVRIG gene) .

In some embodiments, the sequence encoding amino acids 1-163 of mouse PVRIG (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human PVRIG (e.g., amino acids 1-169 of human PVRIG (SEQ ID NO: 2) ) .

In some embodiments, the sequence encoding the entirety or a portion of the extracellular region (with or without the signal peptide) of mouse PVRIG (SEQ ID NO: 1) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding the entirety or a portion of the corresponding region of human PVRIG (SEQ ID NO: 2) . In some embodiments, the corresponding region of human PVRIG comprises the entirety or a portion of the extracellular region (with or without the signal peptide) of human PVRIG. In some embodiments, the sequence encoding amino acids 1-163 of mouse PVRIG (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding a corresponding region of human PVRIG (e.g., amino acids 1-169 of human PVRIG (SEQ ID NO: 2) ) . In some embodiments, the sequence encoding the corresponding region of human PVRIG does not include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids at the N-terminus and/or C-terminus of the extracellular region of human PVRIG.

In some embodiments, the sequence encoding the extracellular domain of mouse PVRIG (SEQ ID NO: 1) is replaced or inactivated. In some embodiments, the sequence is replaced by a sequence encoding the extracellular domain of human PVRIG (SEQ ID NO: 2) . In some embodiments, the sequence encoding amino acids 1-163 of mouse PVRIG (SEQ ID NO: 1) is replaced. In some embodiments, the sequence is replaced by a sequence encoding amino acids 1-169 of human PVRIG (SEQ ID NO: 2) .

In some embodiments, the nucleic acids as described herein are operably linked to a promotor or regulatory element, e.g., an endogenous mouse PVRIG promotor, an inducible promoter, an enhancer, and/or mouse or human regulatory elements.

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are different from a portion of or the entire mouse PVRIG nucleotide sequence (e.g., a portion of exon 1, exon 2 and a portion of exon 3) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire mouse PVRIG nucleotide sequence (e.g., a portion of exon 3, exon 4 and exon 5) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from a portion of or the entire human PVRIG nucleotide sequence (e.g., exon 1, a portion of exon 2, a portion of exon 4, exon 5 and exon 6) .

In some embodiments, the nucleic acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as a portion of or the entire human PVRIG nucleotide sequence (e.g., a portion of exon 2, exon 3, and a portion of exon 4) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire mouse PVRIG amino acid sequence (e.g., amino acids 1-163 of NP_001365367.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire mouse PVRIG amino acid sequence (e.g., amino acids 164-234 of NP_001365367.1 (SEQ ID NO: 1) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from a portion of or the entire human PVRIG amino acid sequence (e.g., amino acids 170-326 of NP_076975.2 (SEQ ID NO: 2) ) .

In some embodiments, the amino acid sequence has at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as a portion of or the entire human PVRIG amino acid sequence (e.g., amino acids 1-169 of NP_076975.2 (SEQ ID NO: 2) ) .

The present disclosure also provides a humanized PVRIG mouse amino acid sequence, wherein the amino acid sequence is selected from the group consisting of:

a) an amino acid sequence shown in SEQ ID NO: 2 or 11;

b) an amino acid sequence having a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

c) an amino acid sequence encoded by a nucleic acid sequence, wherein the nucleic acid sequence is able to hybridize to a nucleotide sequence encoding the amino acid shown in SEQ ID NO: 2 or 11 under a low stringency condition or a strict stringency condition;

d) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

e) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; or

f) an amino acid sequence that comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2 or 11.

The present disclosure also relates to a PVRIG nucleic acid (e.g., DNA or RNA) sequence, wherein the nucleic acid sequence can be selected from the group consisting of:

a) a nucleic acid sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10; a nucleic acid sequence encoding a homologous PVRIG amino acid sequence of a humanized mouse;

b) a nucleic acid sequence that is shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10;

c) a nucleic acid sequence that is able to hybridize to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10 under a low stringency condition or a strict stringency condition;

d) a nucleic acid sequence that has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleotide sequence as shown in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10;

e) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%with or at least 90%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

f) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence has a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%with, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2 or 11;

g) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence is different from the amino acid sequence shown in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and/or

h) a nucleic acid sequence that encodes an amino acid sequence, wherein the amino acid sequence comprises a substitution, a deletion and /or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2 or 11.

The present disclosure also relates to a PVRIG protein sequence, wherein the amino acid sequence of the PVRIG protein can be selected from the group consisting of:

a) all or part of the amino acid sequence shown in SEQ ID NO: 2; or amino acids 1-169 of SEQ ID NO: 2;

b) an amino acid sequence having a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence shown in SEQ ID NO: 2; or amino acids 1-169 of SEQ ID NO: 2;

c) an amino acid sequence that is different from the amino acid sequence shown in SEQ ID NO: 2; or amino acids 1-169 of SEQ ID NO: 2, by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 amino acid; and

d) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 2; or amino acids 1-169 of SEQ ID NO: 2.

The present disclosure also relates to a humanized PVRIG gene sequence, wherein the transcribed mRNA sequence of the humanized PVRIG gene can be selected from the group consisting of:

a) all or part of the nucleotide sequence shown in SEQ ID NO: 10;

b) a nucleotide sequence that at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or at least 99%identical to the nucleotide sequence shown in SEQ ID NO: 10;

c) a nucleotide sequence that is different from the nucleotide sequence shown in SEQ ID NO: 10 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or no more than 1 nucleotide; and

d) a nucleotide sequence that comprises a substitution, a deletion and/or insertion of one or more amino acids to the nucleotide sequence shown at SEQ ID NO: 10.

The disclosure also provides an amino acid sequence that has a homology of at least 90%with, or at least 90%identical to the sequence shown in SEQ ID NO: 2 or 11, and has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 2 or 11 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 2 or 11 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleotide sequence that has a homology of at least 90%, or at least 90%identical to the sequence shown in SEQ ID NO: 10, and encodes a polypeptide that has protein activity. In some embodiments, the homology with the sequence shown in SEQ ID NO: 10 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing homology is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

In some embodiments, the percentage identity with the sequence shown in SEQ ID NO: 10 is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. In some embodiments, the foregoing percentage identity is at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, or 85%.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to any amino acid sequence as described herein. In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded 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 as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of 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. For illustration purposes, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percentage of residues conserved with similar physicochemical properties (percent homology) , e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . The homology percentage, in many cases, is higher than the identity percentage.

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express human or chimeric (e.g., humanized) PVRIG from an endogenous non-human PVRIG locus.

Genetically modified animals

As used herein, the term “genetically-modified non-human animal” refers to a non-human animal having exogenous DNA in at least one chromosome of the animal's genome. In some embodiments, at least one or more cells, e.g., at least 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, 50%of cells of the genetically-modified non-human animal have the exogenous DNA in its genome. The cell having exogenous DNA can be various kinds of cells, e.g., an endogenous cell, a somatic cell, an immune cell, a T cell, a B cell, an antigen presenting cell, a macrophage, a dendritic cell, a germ cell, a blastocyst, or an endogenous tumor cell. In some embodiments, genetically-modified non-human animals are provided that comprise a modified endogenous PVRIG locus that comprises an exogenous sequence (e.g., a human sequence) , e.g., a replacement of one or more non-human sequences with one or more human sequences. The animals are generally able to pass the modification to progeny, i.e., through germline transmission.

As used herein, the term “chimeric gene” or “chimeric nucleic acid” refers to a gene or a nucleic acid, wherein two or more portions of the gene or the nucleic acid are from different species, or at least one of the sequences of the gene or the nucleic acid does not correspond to the wildtype nucleic acid in the animal. In some embodiments, the chimeric gene or chimeric nucleic acid has at least one portion of the sequence that is derived from two or more different sources, e.g., sequences encoding different proteins or sequences encoding the same (or homologous) protein of two or more different species. In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized gene or humanized nucleic acid.

As used herein, the term “chimeric protein” or “chimeric polypeptide” refers to a protein or a polypeptide, wherein two or more portions of the protein or the polypeptide are from different species, or at least one of the sequences of the protein or the polypeptide does not correspond to wild-type amino acid sequence in the animal. In some embodiments, the chimeric protein or the chimeric polypeptide has at least one portion of the sequence that is derived from two or more different sources, e.g., same (or homologous) proteins of different species. In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized protein or a humanized polypeptide.

As used herein, the term “humanized protein” or “humanized polypeptide” refers to a protein or a polypeptide, wherein at least a portion of the protein or the polypeptide is from the human protein or human polypeptide. In some embodiments, the humanized protein or polypeptide is a human protein or polypeptide.

As used herein, the term “humanized nucleic acid” refers to a nucleic acid, wherein at least a portion of the nucleic acid is from the human. In some embodiments, the entire nucleic acid of the humanized nucleic acid is from human. In some embodiments, the humanized nucleic acid is a humanized exon. A humanized exon can be e.g., a human exon or a chimeric exon.

In some embodiments, the chimeric gene or the chimeric nucleic acid is a humanized PVRIG gene or a humanized PVRIG nucleic acid. In some embodiments, at least one or more portions of the gene or the nucleic acid is from the human PVRIG gene, at least one or more portions of the gene or the nucleic acid is from a non-human PVRIG gene. In some embodiments, the gene or the nucleic acid comprises a sequence that encodes a PVRIG protein. The encoded PVRIG protein is functional or has at least one activity of the human PVRIG protein or the non-human PVRIG protein, e.g., binding with human or non-human PVRIG ligand (e.g., PVRL2) ; regulating T cell activation and cytokine production; regulating proliferation of CD4+ and/or CD8+ T cells; enhancing cytotoxic T lymphocyte (CTL) activity; upregulating or downregulating the immune response.

In some embodiments, the chimeric protein or the chimeric polypeptide is a humanized PVRIG protein or a humanized PVRIG polypeptide. In some embodiments, at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a human PVRIG protein, and at least one or more portions of the amino acid sequence of the protein or the polypeptide is from a non-human PVRIG protein. The humanized PVRIG protein or the humanized PVRIG polypeptide is functional or has at least one activity of the human PVRIG protein or the non-human PVRIG protein.

The genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo) , deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey) . For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition) , ” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters) , Cricetidae (e.g., hamster, New World rats and mice, voles) , Muridae (true mice and rats, gerbils, spiny mice, crested rats) , Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice) , Platacanthomyidae (e.g., spiny dormice) , and Spalacidae (e.g., mole rates, bamboo rats, and zokors) . In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae) , a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm) , 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac) , 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999) ; Auerbach et al., Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000) , both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50%BALB/c-50%12954/Sv; or 50%C57BL/6-50%129) .

In some embodiments, the animal is a rodent. In some embodiments, the rodent is selected from BALB/c, A, A/He, A/J, A/WySN, AKR, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2. KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr and C57BL/Ola C57BL, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, CBA/H strains of mice and NOD, NOD/SCID, NOD-Prkdc ^scid IL-2rg ^null Background mice.

In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized PVRIG animal is made. For example, suitable mice for maintaining a xenograft (e.g., a human cancer or tumor) , can have one or more modifications that compromise, inactivate, or destroy the immune system of the non-human animal in whole or in part. Compromise, inactivation, or destruction of the immune system of the non-human animal can include, for example, destruction of hematopoietic cells and/or immune cells by chemical means (e.g., administering a toxin) , physical means (e.g., irradiating the animal) , and/or genetic modification (e.g., knocking out one or more genes) . Non-limiting examples of such mice include, e.g., NOD-Prkdcscid IL-2rγ ^null NOD mice, NOD-Rag 1-/--IL2rg-/- (NRG) mice, Rag 2-/--IL2rg-/- (RG) mice, SCID mice, NOD/SCID mice, IL2Rγ knockout mice, NOD/SCID/γc ^null mice (Ito, M. et al., NOD/SCID/γcnull mouse: an excellent recipient mouse model for engraftment of human cells, Blood 100 (9) : 3175-3182, 2002) , nude mice, and Rag1 and/or Rag2 knockout mice. These mice can optionally be irradiated, or otherwise treated to destroy one or more immune cell type. Thus, in various embodiments, a genetically modified mouse is provided that can include a humanization of at least a portion of an endogenous non-human PVRIG locus, and further comprises a modification that compromises, inactivates, or destroys the immune system (or one or more cell types of the immune system) of the non-human animal in whole or in part. In some embodiments, modification is, e.g., selected from the group consisting of a modification that results in NOD-Prkdcscid IL-2rγ ^null NOD mice, NOD-Rag 1-/--IL2rg-/- (NRG) mice, Rag 2-/--IL2rg-/- (RG) mice, NOD mice, SCID mice, NOD/SCID mice, IL-2Rγ knockout mice, NOD/SCID/γc null mice, nude mice, Rag1 and/or Rag2 knockout mice, and a combination thereof. These genetically modified animals are described, e.g., in US20150106961, which is incorporated herein by reference in its entirety.

In some embodiments, the mouse can include a replacement of all or part of PVRIG coding sequence with human PVRIG coding sequence.

Genetically modified non-human animals that comprise a modification of an endogenous non-human PVRIG locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a PVRIG protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the PVRIG protein sequence) . Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells) , in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous PVRIG locus in the germline of the animal.

Genetically modified animals can express a human PVRIG and/or a chimeric (e.g., humanized) PVRIG from endogenous mouse loci, wherein the endogenous mouse PVRIG gene has been replaced with a human PVRIG gene and/or a nucleotide sequence that encodes a region of human PVRIG sequence or an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70&, 80%, 90%, 95%, 96%, 97%, 98%, or 99%identical to the human PVRIG sequence. In various embodiments, an endogenous non-human PVRIG locus is modified in whole or in part to comprise human nucleic acid sequence encoding at least one protein-coding sequence of a PVRIG protein.

In some embodiments, the genetically modified mice express the human PVRIG and/or chimeric PVRIG (e.g., humanized PVRIG) from endogenous loci that are under control of mouse promoters and/or mouse regulatory elements. The replacement (s) at the endogenous mouse loci provide non-human animals that express human PVRIG or chimeric PVRIG (e.g., humanized PVRIG) in appropriate cell types and in a manner that does not result in the potential pathologies observed in some other transgenic mice known in the art. The human PVRIG or the chimeric PVRIG (e.g., humanized PVRIG) expressed in animal can maintain one or more functions of the wild-type mouse or human PVRIG in the animal. For example, human or non-human PVRIG ligands can bind to the expressed PVRIG, upregulate or downregulate immune response, e.g., upregulate or downregulate immune response by at least 10%, 20%, 30%, 40%, or 50%. Furthermore, in some embodiments, the animal does not express endogenous PVRIG. As used herein, the term “endogenous PVRIG” refers to PVRIG protein that is expressed from an endogenous PVRIG nucleotide sequence of the non-human animal (e.g., mouse) before any genetic modification.

The genome of the animal can comprise a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human PVRIG (NP_076975.2) (SEQ ID NO: 2) . In some embodiments, the genome comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 2.

The genome of the genetically modified animal can comprise a replacement at an endogenous PVRIG gene locus of a sequence encoding a region of endogenous PVRIG with a sequence encoding a corresponding region of human PVRIG. In some embodiments, the sequence that is replaced is any sequence within the endogenous PVRIG gene locus, e.g., exon 1, exon 2, exon 3, exon 4, exon 5, 5'-UTR, 3'-UTR, the first intron, the second intron, and the third intron, the fourth intron, etc. In some embodiments, the sequence that is replaced is within the regulatory region of the endogenous PVRIG gene. In some embodiments, the sequence that is replaced starts within exon 1 and ends within exon 3 of an endogenous mouse PVRIG gene locus.

The genetically modified animal can have one or more cells expressing a human or chimeric PVRIG (e.g., humanized PVRIG) having an extracellular region and a cytoplasmic region, wherein the extracellular region comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, 95%, 99%identical to the extracellular region of human PVRIG. In some embodiments, the extracellular region of the humanized PVRIG has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, or 326 amino acids (e.g., contiguously or non-contiguously) that are identical to human PVRIG. Because human PVRIG and non-human PVRIG (e.g., mouse PVRIG) sequences, in many cases, are different, antibodies that bind to human PVRIG will not necessarily have the same binding affinity with non-human PVRIG or have the same effects to non-human PVRIG. Therefore, the genetically modified animal having a human or a humanized extracellular region can be used to better evaluate the effects of anti-human PVRIG antibodies in an animal model. In some embodiments, the genome of the genetically modified animal comprises a sequence encoding an amino acid sequence that corresponds to part or the entire sequence of exons 2-4 of human PVRIG, part or the entire sequence of the extracellular region of human PVRIG (with or without signal peptide) , or part or the entire sequence of amino acids 1-169 of SEQ ID NO: 2.

In some embodiments, the non-human animal can have, at an endogenous PVRIG gene locus, a nucleotide sequence encoding a chimeric human/non-human PVRIG polypeptide, wherein a human portion of the chimeric human/non-human PVRIG polypeptide comprises a portion of human PVRIG extracellular region, and wherein the animal expresses a functional PVRIG on a surface of a cell of the animal.

In some embodiments, the human portion of the chimeric human/non-human PVRIG polypeptide comprises an amino acid sequence encoded by a portion of exon 2, exon 3, and a portion of exon 4 of human PVRIG. In some embodiments, the human portion of the chimeric human/non-human PVRIG polypeptide comprises a sequence that is at least 80%, 85%, 90%, 95%, or 99%identical to amino acids 1-169 of SEQ ID NO: 2.

In some embodiments, the non-human portion of the chimeric human/non-human PVRIG polypeptide comprises transmembrane and/or cytoplasmic regions of an endogenous non-human PVRIG polypeptide. There may be several advantages that are associated with the transmembrane and/or cytoplasmic regions of an endogenous non-human PVRIG polypeptide. For example, once a PVRIG ligand (e.g., PVRL2) or an anti-PVRIG antibody binds to PVRIG, they can properly transmit extracellular signals into the cells and initiate the downstream pathway. A human or humanized transmembrane and/or cytoplasmic regions may not function properly in non-human animal cells. In some embodiments, a few (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) extracellular amino acids that are close to the transmembrane region of PVRIG are also derived from endogenous sequence. These amino acids can also be important for transmembrane signal transmission. In some embodiments, a few (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids at the N-terminus of the extracellular region are also derived from endogenous sequence.

Furthermore, the genetically modified animal can be heterozygous with respect to the replacement at the endogenous PVRIG locus, or homozygous with respect to the replacement at the endogenous PVRIG locus.

In some embodiments, the humanized PVRIG locus lacks a human PVRIG 5'-UTR. In some embodiments, the humanized PVRIG locus comprises a rodent (e.g., mouse) 5'-UTR. In some embodiments, the humanization comprises a human 3'-UTR. In appropriate cases, it may be reasonable to presume that the mouse and human PVRIG genes appear to be similarly regulated based on the similarity of their 5'-flanking sequence. As shown in the present disclosure, humanized PVRIG mice that comprise a replacement at an endogenous mouse PVRIG locus, which retain mouse regulatory elements but comprise a humanization of PVRIG encoding sequence, do not exhibit pathologies. Both genetically modified mice that are heterozygous or homozygous for humanized PVRIG are grossly normal.

The present disclosure further relates to a non-human mammal generated through the method mentioned above. In some embodiments, the genome thereof contains human gene (s) .

In some embodiments, the non-human mammal is a rodent, and preferably, the non-human mammal is a mouse.

In some embodiments, the non-human mammal expresses a protein encoded by a humanized PVRIG gene.

In addition, the present disclosure also relates to a tumor bearing non-human mammal model, characterized in that the non-human mammal model is obtained through the methods as described herein. In some embodiments, the non-human mammal is a rodent (e.g., a mouse) .

The present disclosure further relates to a cell or cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof, or the tumor bearing non-human mammal; and the tumor tissue derived from the non-human mammal or an offspring thereof when it bears a tumor, or the tumor bearing non-human mammal.

The present disclosure also provides non-human mammals produced by any of the methods described herein. In some embodiments, a non-human mammal is provided; and the genetically modified animal contains the DNA encoding human or humanized PVRIG in the genome of the animal.

In some embodiments, the non-human mammal comprises the genetic construct as described herein (e.g., gene construct as shown in FIG. 2) . In some embodiments, a non-human mammal expressing human or humanized PVRIG is provided. In some embodiments, the tissue-specific expression of human or humanized PVRIG protein is provided.

In some embodiments, the expression of human or humanized PVRIG in a genetically modified animal is controllable, as by the addition of a specific inducer or repressor substance.

Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein. Preferred non-human mammals are mammals, (e.g., rodents) . In some embodiments, the non-human mammal is a mouse.

Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes.

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human PVRIG protein can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies) . In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized PVRIG protein.

Vectors

The present disclosure relates to a targeting vector, comprising: a) a DNA fragment homologous to the 5' end of a region to be altered (5' arm) , which is selected from the PVRIG gene genomic DNAs in the length of 100 to 10,000 nucleotides; b) a desired/donor DNA sequence encoding a donor region; and c) a second DNA fragment homologous to the 3' end of the region to be altered (3' arm) , which is selected from the PVRIG gene genomic DNAs in the length of 100 to 10,000 nucleotides.

In some embodiments, a) the DNA fragment homologous to the 5' end of a conversion region to be altered (5' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000071.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotide sequences that have at least 90%homology to the NCBI accession number NC_000071.7.

In some embodiments, a) the DNA fragment homologous to the 5' end of a region to be altered (5' arm) is selected from the nucleotides from the position 138336113 to the position 138340230 of the NCBI accession number NC_000071.7; c) the DNA fragment homologous to the 3' end of the region to be altered (3' arm) is selected from the nucleotides from the position 138342219 to the position 138345377 of the NCBI accession number NC_000071.7.

In some embodiments, the length of the selected genomic nucleotide sequence in the targeting vector can be about 1 kB, about 1.5 kb, about 2 kb, about 2.5 kb, about 3 kb, about 3.5 kb, about 4 kb, about 4.5 kb, about 5 kb, about 5.5 kb, about 6 kb, about 6.5 kb, about 7 kb, or about 7.5 kb.

In some embodiments, the region to be altered is exon 1, exon 2, exon 3, exon 4, and/or exon 5 of an endogenous PVRIG gene (e.g., a sequence starting within exon 1 and ending within exon 3 of mouse PVRIG gene) .

The targeting vector can further include a selected gene marker.

In some embodiments, the sequence of the 5' arm is shown in SEQ ID NO: 3; and the sequence of the 3' arm is shown in SEQ ID NO: 4. In some embodiments, the donor sequence is shown in SEQ ID NO: 5.

In some embodiments, the donor sequence is derived from human (e.g., 100219911-100220584 of NC_000007.14) . For example, the target region in the targeting vector is a part or entirety of the nucleotide sequence of a human PVRIG, preferably a sequence starting within exon 2 and ending within exon 4 of the human PVRIG. In some embodiments, the nucleotide sequence of the humanized PVRIG encodes the entire or the part of human PVRIG protein with the NCBI accession number NP_076975.2 (SEQ ID NO: 2) .

The disclosure also relates to a cell comprising the targeting vectors as described above.

In addition, the present disclosure further relates to a non-human mammalian cell, having any one of the foregoing targeting vectors, and one or more in vitro transcripts of the construct as described herein. In some embodiments, the cell includes Cas9 mRNA or an in vitro transcript thereof.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

Methods of making genetically modified animals

Genetically modified animals can be made by several techniques that are known in the art, including, e.g., nonhomologous end-joining (NHEJ) , homologous recombination (HR) , zinc finger nucleases (ZFNs) , transcription activator-like effector-based nucleases (TALEN) , and the clustered regularly interspaced short palindromic repeats (CRISPR) -Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., "Delivery technologies for genome editing, " Nature Reviews Drug Discovery 16.6 (2017) : 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

Thus, in some embodiments, the disclosure provides replacing in at least one cell of the animal, at an endogenous PVRIG gene locus, a sequence encoding a region of an endogenous PVRIG with a sequence encoding a corresponding region of human or chimeric PVRIG. In some embodiments, the replacement occurs in a germ cell, a somatic cell, a blastocyst, or a fibroblast, etc. The nucleus of a somatic cell or the fibroblast can be inserted into an enucleated oocyte.

FIG. 3 shows a humanization strategy for a mouse PVRIG locus. In FIG. 3, the targeting strategy involves a vector comprising the 5' end homologous arm, human PVRIG gene fragment, 3' homologous arm. The process can involve replacing endogenous PVRIG sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to replace endogenous PVRIG sequence with human PVRIG sequence.

Thus, in some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous PVRIG locus (or site) , a sequence encoding a region of endogenous PVRIG with a sequence encoding a corresponding region of human PVRIG. The sequence can include a region (e.g., a part or the entire region) of exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6 of a human PVRIG gene. In some embodiments, the sequence encoding a corresponding region of human PVRIG includes a portion of exon 2, exon 3, and a portion of exon 4 of a human PVRIG gene (e.g., a sequence encoding amino acids 1-169 of SEQ ID NO: 2) . In some embodiments, the region is located within the extracellular region of PVRIG.

In some embodiments, the methods of modifying a PVRIG locus of a mouse to express a chimeric human/mouse PVRIG peptide can include the steps of replacing at the endogenous mouse PVRIG locus a nucleotide sequence encoding a mouse PVRIG with a nucleotide sequence encoding a human PVRIG, thereby generating a sequence encoding a chimeric human/mouse PVRIG.

In some embodiments, the nucleotide sequences as described herein do not overlap with each other (e.g., the first nucleotide sequence, the second nucleotide sequence, and/or the third nucleotide sequence do not overlap) . In some embodiments, the amino acid sequences as described herein do not overlap with each other.

The present disclosure further provides a method for establishing a PVRIG gene humanized animal model, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c) .

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57BL/6 mouse) .

In some embodiments, the non-human mammal in step (c) is a female with pseudo pregnancy (or false pregnancy) .

In some embodiments, the fertilized eggs for the methods described above are C57BL/6 fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the methods described above.

Methods of using genetically modified animals

Replacement of non-human genes in a non-human animal with homologous or orthologous human genes or human sequences, at the endogenous non-human locus and under control of endogenous promoters and/or regulatory elements, can result in a non-human animal with qualities and characteristics that may be substantially different from a typical knockout-plus-transgene animal. In the typical knockout-plus-transgene animal, an endogenous locus is removed or damaged and a fully human transgene is inserted into the animal's genome and presumably integrates at random into the genome. Typically, the location of the integrated transgene is unknown; expression of the human protein is measured by transcription of the human gene and/or protein assay and/or functional assay. Inclusion in the human transgene of upstream and/or downstream human sequences are apparently presumed to be sufficient to provide suitable support for expression and/or regulation of the transgene.

In some cases, the transgene with human regulatory elements expresses in a manner that is unphysiological or otherwise unsatisfactory, and can be actually detrimental to the animal. The disclosure demonstrates that a replacement with human sequence at an endogenous locus under control of endogenous regulatory elements provides a physiologically appropriate expression pattern and level that results in a useful humanized animal whose physiology with respect to the replaced gene are meaningful and appropriate in the context of the humanized animal's physiology.

Genetically modified animals that express human or humanized PVRIG protein, e.g., in a physiologically appropriate manner, provide a variety of uses that include, but are not limited to, developing therapeutics for human diseases and disorders, and assessing the toxicity and/or the efficacy of these human therapeutics in the animal models.

In various aspects, genetically modified animals are provided that express human or humanized PVRIG, which are useful for testing agents that can decrease or block the interaction between PVRIG and PVRIG ligands (e.g., PVRL2) or the interaction between PVRIG and anti-human PVRIG antibodies, testing whether an agent can increase or decrease the immune response, and/or determining whether an agent is an PVRIG agonist or antagonist. The genetically modified animals can be, e.g., an animal model of a human disease, e.g., the disease is induced genetically (aknock-in or knockout) . In various embodiments, the genetically modified non-human animals further comprise an impaired immune system, e.g., a non-human animal genetically modified to sustain or maintain a human xenograft, e.g., a human solid tumor or a blood cell tumor (e.g., a lymphocyte tumor, e.g., a B or T cell tumor) .

In some embodiments, the genetically modified animals can be used for determining effectiveness of an anti-PVRIG antibody for the treatment of cancer. The methods involve administering the anti-PVRIG antibody (e.g., anti-human PVRIG antibody) to the animal as described herein, wherein the animal has a tumor; and determining the inhibitory effects of the anti-PVRIG antibody to the tumor. The inhibitory effects that can be determined include, e.g., a decrease of tumor size or tumor volume, a decrease of tumor growth, a reduction of the increase rate of tumor volume in a subject (e.g., as compared to the rate of increase in tumor volume in the same subject prior to treatment or in another subject without such treatment) , a decrease in the risk of developing a metastasis or the risk of developing one or more additional metastasis, an increase of survival rate, and an increase of life expectancy, etc. The tumor volume in a subject can be determined by various methods, e.g., as determined by direct measurement, MRI or CT.

In some embodiments, the tumor comprises one or more cancer cells (e.g., human or mouse cancer cells) that are injected into the animal. In some embodiments, the anti-PVRIG antibody prevents PVRIG ligands from binding to PVRIG. In some embodiments, the anti-PVRIG antibody does not prevent PVRIG ligands from binding to PVRIG.

In some embodiments, the genetically modified animals can be used for determining whether an anti-PVRIG antibody is a PVRIG agonist or antagonist. In some embodiments, the methods as described herein are also designed to determine the effects of the agent (e.g., anti-PVRIG antibodies) on PVRIG, e.g., whether the agent can stimulate immune cells or inhibit immune cells, whether the agent can increase or decrease the production of cytokines, whether the agent can activate or deactivate immune cells, whether the agent can upregulate the immune response or downregulate immune response, and/or whether the agent can induce complement mediated cytotoxicity (CMC) or antibody dependent cellular cytoxicity (ADCC) . In some embodiments, the genetically modified animals can be used for determining the effective dosage of a therapeutic agent for treating a disease in the subject, e.g., cancer, or autoimmune diseases.

The inhibitory effects on tumors can also be determined by methods known in the art, e.g., measuring the tumor volume in the animal, and/or determining tumor (volume) inhibition rate (TGI _TV) . The tumor growth inhibition rate can be calculated using the formula TGI _TV (%) = (1 -TVt/TVc) x 100, where TVt and TVc are the mean tumor volume (or weight) of treated and control groups.

In some embodiments, the anti-PVRIG antibody is designed for treating various cancers. As used herein, the term “cancer” refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term “tumor” as used herein refers to cancerous cells, e.g., a mass of cancerous cells. Cancers that can be treated or diagnosed using the methods described herein include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In some embodiments, the agents described herein are designed for treating or diagnosing a carcinoma in a subject. The term “carcinoma” is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the cancer is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

In some embodiments, the cancer types as described herein include, but not limited to, lymphoma, non-small cell lung cancer (NSCLC) , leukemia, ovarian cancer, nasopharyngeal cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. In some embodiments, the leukemia is selected from acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia. In some embodiments, the lymphoma is selected from Hodgkin's lymphoma and non-Hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T cell lymphoma, and Waldenstrom macroglobulinemia. In some embodiments, the sarcoma is selected from osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma. In some embodiments, the cancer types include head and neck cancer, thyroid cancer, liver cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, glioma, ovarian cancer, stomach cancer, carcinoid, urothelial cancer, breast cancer, endometrial cancer, testis cancer, pancreatic cancer, cervical cancer, renal cancer, lymphoma and skin cancer.

In some embodiments, the antibody is designed for treating various immune disorder or immune-related diseases (e.g., psoriasis, allergic rhinitis, sinusitis, asthma, rheumatoid arthritis, atopic dermatitis, chronic obstructive pulmonary disease (COPD) , chronic bronchitis, emphysema, eczema, osteoarthritis, rheumatoid arthritis, systemic lupus erythematosus, polymyalgia rheumatica, autoimmune hemolytic anemia, systemic vasculitis, pernicious anemia, inflammatory bowel disease, ulcerative colitis, Crohn's disease, or multiple sclerosis) . Thus, the methods as described herein can be used to determine the effectiveness of an anti-PVRIG antibody in inhibiting immune response.

In some embodiments, the immune disorder or immune-related diseases described here include allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, primary thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, self-immune liver disease, diabetes, pain, or neurological disorders.

In some embodiments, the antibody is designed for reducing inflammation (e.g., inflammatory bowel disease, chronic inflammation, asthmatic inflammation, periodontitis, or wound healing) . Thus, the methods as described herein can be used to determine the effectiveness of an antibody for reducing inflammation. In some embodiments, the inflammation described herein includes degenerative inflammation, exudative inflammation, serous inflammation, fibrinitis, suppurative inflammation, hemorrhagic inflammation, necrotitis, catarrhal inflammation, proliferative inflammation, specific inflammation, tuberculosis, syphilis, leprosy, or lymphogranuloma.

In some embodiments, the antibody is designed for treating disorders of bone mineralization, e.g., rickets, renal diseases (renal osteodystrophy, Fanconi syndrome) , tumor-induced osteomalacia, hypophosphatasia, McCune-Albright syndrome, or osteogenesis imperfecta with mineralization defect (syndrome resembling osteogenesis imperfecta (SROI) . In some embodiments, the disorder of bone mineralization is osteoporosis.

The present disclosure also provides methods of determining toxicity of an antibody (e.g., anti-PVRIG antibody) . The methods involve administering the antibody to the animal as described herein. The animal is then evaluated for its weight change, red blood cell count, hematocrit, and/or hemoglobin. In some embodiments, the antibody can decrease the red blood cells (RBC) , hematocrit, or hemoglobin by more than 20%, 30%, 40%, or 50%. In some embodiments, the animals can have a weight that is at least 5%, 10%, 20%, 30%, or 40%smaller than the weight of the control group (e.g., average weight of the animals that are not treated with the antibody) .

The present disclosure also relates to the use of the animal model generated through the methods as described herein in the development of a product related to an immunization processes of human cells, the manufacturing of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

In some embodiments, the disclosure provides the use of the animal model generated through the methods as described herein in the production and utilization of an animal experimental disease model of an immunization processes involving human cells, the study on a pathogen, or the development of a new diagnostic strategy and/or a therapeutic strategy.

The disclosure also relates to the use of the animal model generated through the methods as described herein in the screening, verifying, evaluating or studying the PVRIG gene function, human PVRIG antibodies, drugs for human PVRIG targeting sites, the drugs or efficacies for human PVRIG targeting sites, the drugs for immune-related diseases and antitumor drugs.

In some embodiments, the disclosure provides a method to verify in vivo efficacy of TCR-T, CAR-T, and/or other immunotherapies (e.g., T-cell adoptive transfer therapies) . For example, the methods include transplanting human tumor cells into the animal described herein, and applying human CAR-T to the animal with human tumor cells. Effectiveness of the CAR-T therapy can be determined and evaluated. In some embodiments, the animal is selected from the PVRIG gene humanized non-human animal prepared by the methods described herein, the PVRIG gene humanized non-human animal described herein, the double-or multi-humanized non-human animal generated by the methods described herein (or progeny thereof) , a non-human animal expressing the human or humanized PVRIG protein, or the tumor-bearing or inflammatory animal models described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies can treat the PVRIG-associated diseases described herein. In some embodiments, the TCR-T, CAR-T, and/or other immunotherapies provides an evaluation method for treating the PVRIG-associated diseases described herein.

Genetically modified animal model with two or more human or chimeric genes

The present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes. The animal can comprise a human or chimeric PVRIG gene and a sequence encoding an additional human or chimeric protein.

In some embodiments, the additional human or chimeric protein can be T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) , cytotoxic T-lymphocyte-associated protein 4 (CTLA4) , DNAM-1 (CD226) , programmed cell death protein 1 (PD-1) , IL4, Colony Stimulating Factor 1 (CSF1) , IL34, C-C Motif Chemokine Receptor 2 (CCR2) , CD40, C-X-C Motif Chemokine Receptor 4 (CXCR4) , Vascular Endothelial Growth Factor (VEGF) , cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) , Lymphocyte Activating 3 (LAG-3) , B And T Lymphocyte Associated (BTLA) , Programmed Cell Death 1 Ligand 1 (PD-L1) , CD27, CD28, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) , T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) , Glucocorticoid-Induced TNFR-Related Protein (GITR) , Signal regulatory protein α (SIRPα) or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40) .

The methods of generating genetically modified animal model with two or more human or chimeric genes (e.g., humanized genes) can include the following steps:

(a) using the methods of introducing human PVRIG gene or chimeric PVRIG gene as described herein to obtain a genetically modified non-human animal;

(b) breeding the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPα, CTLA4, CD226, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, BTLA, CD28, CD137, CD154, or GITR. Some of these genetically modified non-human animal are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2017/110494, PCT/CN2017/110435, PCT/CN2017/120388, PCT/CN2018/081628, PCT/CN2018/081629; each of which is incorporated herein by reference in its entirety.

In some embodiments, the PVRIG gene humanization is directly performed on a genetically modified animal having a human or chimeric PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40 gene. In some embodiments, the genetically modified animal has a human or chimeric PD-1. In some embodiments, the genetically modified animal has a human or chimeric TIGIT. In some embodiments, the genetically modified animal has a human or chimeric PD-1 and TIGIT.

As these proteins may involve different mechanisms, a combination therapy that targets two or more of these proteins thereof may be a more effective treatment. In fact, many related clinical trials are in progress and have shown a good effect. The genetically modified animal model with two or more human or humanized genes can be used for determining effectiveness of a combination therapy that targets two or more of these proteins, e.g., an anti-PVRIG antibody and an additional therapeutic agent (e.g., an anti-PD-1 antibody) for the treatment of cancer. The methods include administering the anti-PVRIG antibody and the additional therapeutic agent (e.g., an anti-PD-1 antibody) to the animal, wherein the animal has a tumor; and determining the inhibitory effects of the combined treatment to the tumor. In some embodiments, the additional therapeutic agent is an antibody that specifically binds to PD-1, IL4, CSF1, IL34, CCR2, CD40, CXCR4, VEGF, CTLA-4, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPα, or OX40. In some embodiments, the additional therapeutic agent is an anti-CTLA4 antibody (e.g., ipilimumab) , an anti-PD-1 antibody (e.g., nivolumab) , or an anti-PD-L1 antibody.

In some embodiments, the animal further comprises a sequence encoding a human or humanized PD-1, a sequence encoding a human or humanized PD-L1, or a sequence encoding a human or humanized CTLA-4. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab) , an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the tumor comprises one or more tumor cells that express CD80, CD86, PD-L1, and/or PD-L2. In some embodiments, the tumor comprises one or more tumor cells that express PVRL2 and/or PD-L1.

In some embodiments, the combination treatment is designed for treating various cancer as described herein, e.g., melanoma, non-small cell lung carcinoma (NSCLC) , small cell lung cancer (SCLC) , bladder cancer, prostate cancer (e.g., metastatic hormone-refractory prostate cancer) , advanced breast cancer, advanced ovarian cancer, and/or advanced refractory solid tumor. In some embodiments, the combination treatment is designed for treating metastatic solid tumors, NSCLC, melanoma, B-cell non-Hodgkin lymphoma, colorectal cancer, and multiple myeloma. In some embodiments, the combination treatment is designed for treating melanoma, carcinomas (e.g., pancreatic carcinoma) , mesothelioma, hematological malignancies (e.g., Non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia) , or solid tumors (e.g., advanced solid tumors) . In some embodiments, the combination treatment is designed for treating liver cancer, pancreatic cancer, osteosarcoma, breast cancer, ovarian cancer, endometrial cancer, oral squamous cell carcinoma, cervical cancer, renal cancer, head and neck cancer, or brain cancer.

In some embodiments, the methods described herein can be used to evaluate the combination treatment with some other methods. The methods of treating a cancer that can be used alone or in combination with methods described herein, include, e.g., treating the subject with chemotherapy, e.g., campothecin, doxorubicin, cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-flurouracil, vincristin, vinblastin, and/or methotrexate. Alternatively or in addition, the methods can include performing surgery on the subject to remove at least a portion of the cancer, e.g., to remove a portion of or all of a tumor (s) , from the patient.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials were used in the following examples.

SacI, EcoNI, and NdeI enzymes were purchased from NEB, and the product numbers are R3156V, R0521V, and R0111V, respectively;

C57BL/6 mice and Flp tool mice were purchased from the National Rodent Laboratory Animal Seed Center of China National Academy of Food and Drug Control;

Brilliant Violet 510 ^TM anti-mouse CD45 was purchased from Biolegend, Cat. No. 103138;

PE anti-human PVRIG Antibody was purchased from R&D, catalog number MAB93651;

Purified anti-mouse CD16/32 was purchased from Biolegend, Cat. No. 101302.

EXAMPLE 1: PVRIG gene humanized mouse

Mouse PVRIG gene (NCBI Gene ID: 102640920, Primary source: MGI: 5596028, UniProt: A0A1B0GS01, located at positions 138340193 to 138346953 of chromosome 5 (NC_000071.7) , based on transcript NM_001378438.1 and its encoded protein NP_001365367.1 (SEQ ID NO: 1) ) and human PVRIG gene (NCBI Gene ID: 79037, Primary source: HGNC: 32190, UniProt ID: Q6DKI7, located at positions 100218625 to 100221490 of chromosome 7 (NC_000007.14) , based on transcript NM_024070.3 and its encoded protein NP_076975.2 (SEQ ID NO: 2) ) were used. A schematic diagram of the comparison between mouse PVRIG gene and human PVRIG gene is shown in FIG. 1.

To prepare the PVRIG humanized mice, a nucleotide sequence encoding human PVRIG protein was introduced into the mouse endogenous PVRIG locus, so that the mouse expresses a human or humanized PVRIG protein. Specifically, using gene editing technology, under the control of the endogenous regulatory elements of the mouse PVRIG gene, the coding region of the mouse PVRIG gene was replaced with the coding region of the human PVRIG gene. Alternatively, a sequence containing a part of exon 2, exon 3, and a part of exon 4 of the human PVRIG gene (about 0.65kb) was used to replace a sequence containing a part of exon 1, exon 2 and a part of exon 3 of the mouse PVRIG gene (about 0.65kb) . A schematic diagram of the humanized PVRIG locus is shown in FIG. 2.

A targeting strategy is shown in FIG. 3. The targeting vector contains the homology arm sequences upstream and downstream of the replaced region in the mouse PVRIG gene, and an A fragment containing the human PVRIG sequence. Among them, the upstream homology arm sequence (5′homology arm, SEQ ID NO: 3) is the same as the nucleotide sequence from 138336113 to 138340230 of the NCBI accession number NC_000071.7, and the downstream homology arm sequence (3′homology arm, SEQ ID NO: 4) is identical to the nucleotide sequence at positions 138342219 to 138345377 of NCBI Accession No. NC_000071.7. The nucleotide sequence of human PVRIG on the A fragment (SEQ ID NO: 5) is the same as the nucleotide sequence from 100219911 to 100220584 of the NCBI accession number NC_000007.14. The upstream connection between the human PVRIG sequence and the mouse sequence is designed as:

where the last “C” in the sequence

is the last nucleotide in the mouse sequence, and the first “A” in the sequence “ ATGA” is the first nucleotide in the human sequence. The downstream connection between the human PVRIG sequence and the mouse is designed as

where the last “G” in the sequence

is the last nucleotide in the human sequence, and the first “G” in the sequence “ GCAG” is the first nucleotide of the mouse sequence.

The targeting vector also includes a resistance gene for positive clone selection (positive selection marker) , namely the coding sequence neomycin phosphotransferase (Neo) , which is flanked by two site-specific recombination sites (Frt) arranged in the same direction, forming a Neo cassette. The connection between the 5′end of the Neo cassette and the mouse sequence is designed as

where "G" in the sequence

is the last nucleotide of the mouse sequence, and the first "C" in the sequence “ CCTA" is the first nucleotide of the Neo cassette. The connection between the 3′end of the Neo cassette and the mouse gene is designed as

where the "G" in the sequence

is the last nucleotide of the Neo cassette, and the first "C" in the sequence " CTTC" is the first nucleotide of the mouse sequence. In addition, a negative selection marker (the coding gene for diphtheria toxin A subunit (DTA) ) was also included downstream of the 3′homology arm of the targeting vector. The mRNA sequence of the humanized mouse PVRIG gene is shown in SEQ ID NO: 10, and the expressed protein sequence is shown in SEQ ID NO: 11.

Given that human PVRIG has multiple isoforms or transcripts, the methods described herein can be applied to other isoforms or transcripts.

The construction of the targeting vector can be carried out by enzyme cleavage and ligation. The constructed targeting vector was preliminarily verified by enzyme digestion, and then sent to a sequencing company for sequencing verification. The targeted vector verified by sequencing was electroporated into embryonic stem cells of C57BL/6 mice, and the obtained cells were screened with positive clone selection marker. PCR and Southern Blot technology were used to confirm the integration of the exogenous sequence. Specifically, the correct positive clones were screened out, the positive clones were identified by PCR, and then Southern Blot was performed (cellular DNA was digested with SacI, EcoNI, and NdeI enzymes, and 3 probes were used for hybridization, as shown in the table below) . The detection results are shown in FIG. 4. As shown in FIG. 4 and verified by sequencing, 1-B08, 3-E06, 3-F11 and 3-G06 are all positive clones without random insertion.

Table 3: Southern blot probes and target fragment lengths

The PCR assay includes the following primers:

WT-F: 5'-GTGTAGGGCTGAATGGGATAAGGGC-3' (SEQ ID NO: 12) ,

Mut-R: 5'-GTGACACACAAGGTCAGCAGCAC -3' (SEQ ID NO: 13) ;

The Southern Blot detection includes the following probe primers:

5'Probe:

5'Probe-F: 5'-AAGGCCCCTCCATAGACTTCTCAGC -3' (SEQ ID NO: 14) ,

5'Probe-R: 5'-GGGGCTGCTAACGTCAATCACCTAC -3' (SEQ ID NO: 15) ;

3'Probe:

3'Probe-F: 5'-TCCATGGAACGTTGAATACCAGAAGT -3' (SEQ ID NO: 16) ,

3'Probe-R: 5'-TGGTGCAAGTCTATAATTACAGCAGGTC -3' (SEQ ID NO: 17) ;

Neo Probe:

Neo Probe -F: 5'-GGATCGGCCATTGAACAAGAT -3' (SEQ ID NO: 18) ,

Neo Probe -R: 5'-CAGAAGAACTCGTCAAGAAGGC -3' (SEQ ID NO: 19) .

The positive clones that had been screened (black mice) were introduced into isolated blastocysts (white mice) , and the chimeric blastocysts were transferred to a culture medium for short-term culture and then transplanted to the fallopian tubes of the recipient mother (white mice) to produce the F0 chimeric mice (black and white) . The F2 generation homozygous mice were obtained by backcrossing the F0 generation chimeric mice with wild-type mice to obtain the F1 generation mice, and then mating the F1 generation heterozygous mice with each other. The positive mice can also be mated with the Flp transgenic mice to remove the positive selectable marker gene (FIG. 5) , and then mated with each other to obtain the humanized PVRIG homozygous mice. The genotype of the progeny mice can be identified by PCR using primers shown in the table below. The identification results of exemplary F1 generation mice (Neo cassette-removed) are shown in FIGS. 6A-6D, and mice labelled F1-01, F1-02, F1-03 were identified as positive heterozygous mice.

Table 4: Primer names and sequences

The above results indicated that this method can be used to construct genetically engineered PVRIG mice and the genetic modification can be stably passed to the next generation without random insertions.

EXAMPLE 2 Detection of protein expression in PVRIG humanized mice

The expression of humanized PVRIG mRNA in positive mice was determined by conventional methods, such as RT-PCR. Specifically, a 5-week-old female C57BL/6 wild-type mouse and a PVRIG gene humanized homozygous mouse prepared in Example 1 were taken, and the spleen tissues were collected after euthanasia (cervical dislocation) . Cellular RNA was extracted using the Trizol kit (according to manufacturer instructions) , reverse transcribed into cDNA, and then detected by RT-PCR. The detection results are shown in FIGS. 7A-7C. As can be seen from FIGS. 7A-7C, in the spleen of C57BL/6 wild-type mice, murine PVRIG mRNA was detected, and no humanized PVRIG mRNA was detected. By contrast, in the spleen of PVRIG humanized homozygous mice, only humanized PVRIG mRNA was detected, and no mouse PVRIG mRNA was detected.

RT-PCR primer sequences include:

PCR-F1: 5'-TGACCTTGTGTGTCACTGCG -3' (SEQ ID NO: 25)

PCR-R1: 5'-GGCTGAGATTTACTCAAACACCA-3' (SEQ ID NO: 26)

PCR-F2: 5'-GTCTCTGAAGCAAGCCCTGAG-3' (SEQ ID NO: 27)

PCR-R2: 5'-GGTGCTAGTGAGAGATGGCTG -3' (SEQ ID NO: 28)

GAPDH-F: 5'-TCACCATCTTCCAGGAGCGAGA-3' (SEQ ID NO: 29)

GAPDH-R: 5'-GAAGGCCATGCCAGTGAGCTT-3' (SEQ ID NO: 30)

The expression of humanized PVRIG protein in positive mice was confirmed by conventional methods, such as flow cytometry. Specifically, a 5-week-old female C57BL/6 wild-type mouse and a 5-week-old female PVRIG humanized homozygous mouse were taken, and the spleen tissues were collected after euthanasia (cervical dislocation) . The cells were stained with PE Anti-human PVRIG Antibody (hPVRIG) , Brilliant Violet 510 ^TM anti-mouse CD45 (mCD45) , and Purified anti-mouse CD16/32, and analyzed by flow cytometry. The results showed that, Immune cells (characterized as mCD45+) in the spleen of C57BL/6 mice contained 0.99%hPVRIG-positive cells (characterized as mCD45+hPVRIG+) . Immune cells in the spleen of PVRIG humanized homozygous mice had 30.3%hPVRIG positive cells (mCD45+hPVRIG+) . The results indicated that the spleen cells of C57BL/6 mice did not express the humanized PVRIG protein, and the PVRIG humanized mouse spleen cells expressed the humanized PVRIG protein.

The immunophenotyping of leukocytes and T cells in the spleen, peripheral blood and lymph nodes of wild-type C57BL/6 mice (C57BL/6) and PVRIG humanized homozygous mice (B-hPVRIG) was further detected by flow cytometry. The detection results of leukocyte subtypes and T cell subtypes in the spleen are shown in FIG. 8 and FIG. 9, respectively, and the detection results of leukocyte subtypes and T cell subtypes in peripheral blood are shown in FIG. 10 and FIG. 11, respectively. As can be seen from the figures, in the spleen samples of PVRIG gene humanized homozygous mice, the percentages of B cells, T cells, NK cells, CD4+ T cells, CD8+ T cells, granulocytes, dendritic cells, macrophages, monocytes and other leukocyte subtypes were basically the same as the percentages in C57BL/6 wild type mice (FIG. 8 and FIG. 9) . Similarly, in the peripheral blood samples of PVRIG gene humanized homozygous mice, the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells, and Tregs cells were basically the same as the percentages in C57BL/6 wild-type mice (FIG. 10 and FIG. 11) .

The flow cytometry detection results of leukocyte subtypes and T cell subtypes in the lymph nodes are shown in FIG. 12 and FIG. 13, respectively. As shown in the figures, in the PVRIG humanized homozygous mouse lymph node samples, the percentages of leukocyte subtypes such as B cells, T cells, NK cells, CD4+ T cells and CD8+ T cells were basically the same as the percentages in C57BL/6 wild-type mice (FIG. 12) . Similarly, in the PVRIG humanized homozygous mouse lymph node samples, the percentages of T cell subtypes such as CD4+ T cells, CD8+ T cells and Tregs cells were basically the same as the percentages in C57BL/6 wild-type mice (FIG. 13) .

EXAMPLE 3: Generation of double-or multi-gene humanized mice

The humanized PVRIG mouse prepared by the methods described herein can also be used to prepare a double-or multi-gene humanized mouse model. For example, in Example 1, the embryonic stem cells used for blastocyst microinjection can be selected from mice containing genetic modifications, e.g., PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPα, CTLA4 and/or CD226 gene humanized mice, thereby generating double-gene humanized mice. Alternatively, the embryonic stem cells of PVRIG gene humanized mice can be selected for gene editing, to obtain a double-gene or multi-gene humanized mouse model comprising humanized PVRIG and other genetic modifications. In addition, it is also possible to breed the homozygous or heterozygous PVRIG transgenic mice obtained by the methods described herein with other genetically modified homozygous or heterozygous mice, and the offspring can be screened. According to Mendel's law, it is possible to generate double-gene or multi-gene modified heterozygous mice comprising humanized PVRIG gene and other genetic modifications. Then the heterozygous mice can be bred with each other to obtain homozygous double-gene or multi-gene humanized mice.

A similar method can also be used for the generation of triple-gene humanized mice. For example, two single-gene humanized mice can be bred to generate double-gene humanized mice, and then the double-gene humanized mice can be bred with another single-gene humanized mice. Positive offspring can be screened to generate triple-gene humanized mice.

EXAMPLE 3. In vivo efficacy verification

The PVRIG gene humanized mice prepared by the invention were used to construct tumor models to test the efficacy of drugs targeting human PVRIG. Specifically, the PVRIG gene humanized homozygous mice (6 weeks old) prepared in Example 1 were selected and subcutaneously inoculated with mouse colon cancer cells MC38 (5×10 ⁵ cells) . When the tumor volume reached about 100-150 mm ³, the mice were divided into control group or treatment group (5 mice per group) based on tumor volume. The control group received via injection hIgG (purchased from: Beijing Dingguo Changsheng Biotechnology Co., Ltd.; product number: AG-0011) , and the treatment group received via injection COM-701 (G2, the drug information can be found in patent US10213505B2) and SRF-813 antibody (G3, the drug information can be found in patent Publication No. US20200040081A1) ) . The specific group assignment and dosage are shown in the table below. Tumor volume was measured twice a week and the body weight of the mice was measured as well. Euthanasia was performed when the tumor volume of the mouse reached 3000 mm ³. The body weight and tumor volume measurement results of mice during the experimental period are shown in Figures 14-16, respectively.

Table 5: Group assignment and dosage

The results of the in vivo efficacy verification experiment are listed in the table below. The results include tumor volume at the time of group assignment, 14 days after group assignment, and 17 days after group assignment, mouse survival, tumor-free mice, tumor (volume) inhibition Tumor Growth Inhibition value (TGITV) and the statistical difference (P value) of tumor volume between the treatment group and the control group.

Table 6: Tumor volume, survival and tumor inhibition rate

As shown in FIGS. 14, 15 and Table 6, overall, the mice in each group were in good health during the experiment. Comparing to the control group (G1) , the treatment group (G2 and G3) showed an increasing trend in body weight (FIGS. 14 and 15) , and there was no significant difference (P＞0.05) in body weight, indicating that human PVRIG antibodies COM-701 and SRF-813 were well tolerated, and did not produce obvious toxic effects. As shown in FIG. 16 and Table 6, the tumor volumes of the treatment group were smaller than those of the control group at each stage. On the 17th day, the tumor volumes of the mice in the G2 and G3 groups were 1662 ± 312 mm ³ and 2058 ± 433 mm ³, both smaller than the tumor volume of the control group (2388 ± 243mm ³) . The results showed that the PVRIG humanized mice prepared by the disclosed method can be used for the in vivo efficacy verification of drugs targeting human PVRIG.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

A genetically-modified, non-human animal whose genome comprises at least one chromosome comprising a sequence encoding a human or chimeric PVRIG.
The animal of claim 1, wherein the sequence encoding the human or chimeric PVRIG is operably linked to an endogenous regulatory element at the endogenous PVRIG gene locus in the at least one chromosome.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric PVRIG comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to human PVRIG (NP_076975.2 (SEQ ID NO: 2) ) .
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric PVRIG comprises a sequence encoding an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to SEQ ID NO: 11.
The animal of claim 1 or 2, wherein the sequence encoding a human or chimeric PVRIG comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to amino acids 1-169 of SEQ ID NO: 2.
The animal of any one of claims 1-5, wherein the animal is a mammal, e.g., a monkey, a rodent, or a mouse.
The animal of any one of claims 1-6, wherein the animal is a mouse.
The animal of any one of claims 1-7, wherein the animal does not express endogenous PVRIG.
The animal of any one of claims 1-8, wherein the animal has one or more cells expressing human or chimeric PVRIG.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric PVRIG, and a human PVRIG ligand can bind to the expressed human or chimeric PVRIG.
The animal of any one of claims 1-9, wherein the animal has one or more cells expressing human or chimeric PVRIG, and an endogenous PVRIG ligand can bind to the expressed human or chimeric PVRIG.
A genetically-modified, non-human animal, wherein the genome of the animal comprises a replacement of a sequence encoding a region of endogenous PVRIG with a sequence encoding a corresponding region of human PVRIG at an endogenous PVRIG gene locus.
The animal of claim 12, wherein the sequence encoding the corresponding region of human PVRIG is operably linked to an endogenous regulatory element at the endogenous PVRIG locus, and one or more cells of the animal expresses a chimeric PVRIG.
The animal of claim 12 or 13, wherein the animal does not express endogenous PVRIG.
The animal of any one of claims 12-14, wherein the sequence encodes all or a portion of the extracellular region (with or without signal peptide) of endogenous PVRIG is replaced with all or a portion of the extracellular region (with or without signal peptide) of human PVRIG.
The animal of any one of claims 12-15, wherein the animal has one or more cells expressing a chimeric PVRIG having a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region.
The animal of claim 16, wherein the chimeric PVRIG has a sequence that has at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 contiguous amino acids that are identical to a contiguous sequence present in human PVRIG.
The animal of any one of claims 12-17, wherein the sequence encoding a region of endogenous PVRIG comprises exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous PVRIG gene.
The animal of claim 18, wherein the animal is a mouse, and the sequence encoding a region of endogenous PVRIG starts within exon 1 and ends within exon 3 of the endogenous mouse PVRIG gene.
The animal of any one of claims 12-19, wherein the animal is heterozygous with respect to the replacement at the endogenous PVRIG gene locus.
The animal of any one of claims 12-19, wherein the animal is homozygous with respect to the replacement at the endogenous PVRIG gene locus.
A method for making a genetically-modified, non-human animal, comprising:

replacing in at least one cell of the animal, at an endogenous PVRIG gene locus, a sequence encoding a region of an endogenous PVRIG with a sequence encoding a corresponding region of human PVRIG.
The method of claim 22, wherein the sequence encoding the corresponding region of human PVRIG comprises exon 1, exon 2, exon 3, exon 4, exon 5, and/or exon 6, or a part thereof, of a human PVRIG gene.
The method of claim 22 or 23, wherein the sequence encoding the corresponding region of human PVRIG starts within exon 2 and ends within exon 4 of a human PVRIG gene.
The method of any one of claims 22-24, wherein the sequence encoding the corresponding region of human PVRIG encodes amino acids 1-169 of SEQ ID NO: 2.
The method of any one of claims 22-25, wherein the replaced region of an endogenous PVRIG is located within the extracellular region.
The method of any one of claims 22-26, wherein the sequence encoding a region of endogenous PVRIG comprises exon 1, exon 2, exon 3, exon 4, and/or exon 5, or a part thereof, of the endogenous PVRIG gene.
The method of claim 27, wherein the animal is a mouse, and the replaced sequence of an endogenous PVRIG starts within exon 1 and ends within exon 3 of the endogenous mouse PVRIG gene.
A non-human animal comprising at least one cell comprising a nucleotide sequence encoding a chimeric PVRIG polypeptide, wherein the chimeric PVRIG polypeptide comprises at least 50 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human PVRIG, wherein the animal expresses the chimeric PVRIG.
The animal of claim 29, wherein the chimeric PVRIG polypeptide has at least 50, at least 100, or at least 150 contiguous amino acid residues that are identical to the corresponding contiguous amino acid sequence of a human PVRIG.
The animal of claim 29 or 30, wherein the chimeric PVRIG polypeptide comprises a sequence that is at least 90%, 95%, or 99%identical to amino acids 1-169 of SEQ ID NO: 2.
The animal of any one of claims 29-31, wherein the nucleotide sequence is operably linked to an endogenous PVRIG regulatory element of the animal.
The animal of any one of claims 29-32, wherein the chimeric PVRIG polypeptide comprises an endogenous PVRIG transmembrane region and/or an endogenous PVRIG cytoplasmic region.
The animal of any one of claims 29-33, wherein the nucleotide sequence is integrated to an endogenous PVRIG gene locus of the animal.
The animal of any one of claims 29-34, wherein the chimeric PVRIG has at least one mouse PVRIG activity and/or at least one human PVRIG activity.
A method of making a genetically-modified non-human animal cell that expresses a chimeric PVRIG, the method comprising:

replacing at an endogenous PVRIG gene locus, a nucleotide sequence encoding a region of endogenous PVRIG with a nucleotide sequence encoding a corresponding region of human PVRIG, thereby generating a genetically-modified non-human animal cell that includes a nucleotide sequence that encodes the chimeric PVRIG, wherein the non-human animal cell expresses the chimeric PVRIG.
The method of claim 36, wherein the animal is a mammal, e.g., a monkey, a rodent, or a mouse.
The method of claim 36 or 37, wherein the chimeric PVRIG comprises a human or humanized extracellular region, an endogenous transmembrane region, and an endogenous cytoplasmic region.
The method of any one of claims 36-38, wherein the nucleotide sequence encoding the chimeric PVRIG is operably linked to an endogenous PVRIG regulatory region, e.g., promoter.
The animal of any one of claims 1-21 and 29-35, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The animal of claim 40, wherein the additional human or chimeric protein is PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPα, CTLA4, and/or CD226.
The animal of claim 40, wherein the additional human or chimeric protein is PD-1 and/or TIGIT, and the animal expresses the human or chimeric PD-1 and/or TIGIT.
The method of any one of claims 22-28 and 36-39, wherein the animal further comprises a sequence encoding an additional human or chimeric protein.
The method of claim 43, wherein the additional human or chimeric protein is PD-1, PD-L1, TIGIT, OX40, LAG3, TIM3, CD27, CD47, SIRPα, CTLA4, or CD226.
The method of claim 43, wherein the additional human or chimeric protein is PD-1 and/or TIGIT, and the animal expresses the human or chimeric PD-1 and/or TIGIT.
A method of determining effectiveness of an anti-PVRIG antibody for the treatment of cancer, comprising:

administering the anti-PVRIG antibody to the animal of any one of claims 1-21, 29-35, or 40-42, wherein the animal has a cancer; and

determining the inhibitory effects of the anti-PVRIG antibody to the cancer.
The method of claim 46, wherein one or more cells (e.g., T or NK cells) in the cancer express PVRIG.
The method of claim 46 or 47, wherein the cancer comprises one or more cancer cells that are injected into the animal.
The method of any one of claims 46-48, wherein determining the inhibitory effects of the anti-PVRIG antibody to the cancer involves measuring the tumor volume in the animal.
The method of any one of claims 46-49, wherein the cancer is head and neck cancer, thyroid cancer, liver cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, glioma, ovarian cancer, stomach cancer, carcinoid, urothelial cancer, breast cancer, endometrial cancer, testis cancer, pancreatic cancer, cervical cancer, renal cancer, lymphoma or skin cancer.
A method of determining effectiveness of an anti-PVRIG antibody and an additional therapeutic agent for the treatment of cancer, comprising

administering the anti-PVRIG antibody and the additional therapeutic agent to the animal of any one of claims 1-21, 29-35, or 40-42, wherein the animal has a cancer; and

determining the inhibitory effects on the cancer.
The method of claim 51, wherein the animal further comprises a sequence encoding a human or chimeric programmed cell death protein 1 (PD-1) .
The method of claim 51 or 52, wherein the animal further comprises a sequence encoding a human or chimeric TIGIT.
The method of any one of claims 51-53, wherein the additional therapeutic agent is an anti-PD-1 antibody or an anti-TIGIT antibody.
The method of claim any one of claims 51-54, wherein the cancer comprises one or more cancer cells that express a PVRIG ligand (e.g., PVRL2) .
The method of any one of claims 51-55, wherein the cancer is caused by injection of one or more cancer cells into the animal.
The method of any one of claims 51-56, wherein determining the inhibitory effects of the treatment involves measuring the tumor volume in the animal.
The method of any one of claims 51-57, wherein the animal has head and neck cancer, thyroid cancer, liver cancer, prostate cancer, lung cancer, melanoma, colorectal cancer, glioma, ovarian cancer, stomach cancer, carcinoid, urothelial cancer, breast cancer, endometrial cancer, testis cancer, pancreatic cancer, cervical cancer, renal cancer, lymphoma or skin cancer.
A protein comprising an amino acid sequence, wherein the amino acid sequence is one of the following:

(a) an amino acid sequence set forth in SEQ ID NO: 2 or 11;

(b) an amino acid sequence that is at least 90%identical to SEQ ID NO: 2 or 11;

(c) an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 2 or 11;

(d) an amino acid sequence that is different from the amino acid sequence set forth in SEQ ID NO: 2 or 11 by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid; and

(e) an amino acid sequence that comprises a substitution, a deletion and/or insertion of one, two, three, four, five or more amino acids to the amino acid sequence set forth in SEQ ID NO: 2 or 11.
A nucleic acid comprising a nucleotide sequence, wherein the nucleotide sequence is one of the following:

(a) a sequence that encodes the protein of claim 59;

(b) SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10;

(c) a sequence that is at least 90%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10; and

(d) a sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, or 10.
A cell comprising the protein of claim 59 and/or the nucleic acid of claim 60.
An animal comprising the protein of claim 59 and/or the nucleic acid of claim 60.