Engineered red blood cell for site-directed conjugation of protein by means of chemical small molecule linker

Abstract

A targeted red blood cell. The present invention specifically relates to a protein drug molecule-red blood cell conjugate, and more specifically relates to an engineered red blood cell carrying an antibody (for example, an antibody directed against PD-1 or PD-L1), a preparation method therefor, and a method and the use thereof for treating cancer, in particular a cancer insensitive or tolerant to the antibody.

Description

An engineered red blood cell by site-specific coupling of proteins with a small molecule linker Technical Field

[0001] The present invention relates to a method for targeting erythrocytes, specifically to a protein drug molecule-erythrocyte conjugate, more specifically to engineered erythrocytes carrying antibodies (e.g., antibodies against PD-1 or PD-L1), a preparation method thereof, and methods and uses thereof for treating cancer, particularly cancers that are insensitive or tolerant to the antibodies. Background technology:

[0002] The spleen is a filter for blood circulation. Numerous studies have shown that red blood cells in the body are generally cleared in the spleen to complete the normal metabolism of red blood cells (Structure and function of the immune system in the spleen. Nat Rev Immunol. 2005Aug;5(8):606-16;The spleen in local and systemic regulation of immunity. Immunity. 2013Nov 14;39(5):806-18). Therefore, red blood cells are considered to be a good transport carrier for targeting the spleen, which can achieve the targeted delivery of immune drugs. The Harvard University research team used red blood cell-driven immune targeting, carried OVA antigen, as a therapeutic vaccine to activate specific T cells, thereby successfully achieving tumor regression in a mouse tumor model (Erythrocyte-driven immunization via biomimicry of their natural antigen-presenting function. Samir Mitragotri et al. Proc Natl Acad Sci USA 2020Jul 28;117(30):17727-17736.). Studies have shown that drugs carried on cells usually achieve better drug-cell interactions (Membrane interactions in drug delivery: Model cell membranes and orthogonal techniques. Adv Colloid Interface Sci. 2020 Jul; 281: 102177; Drug delivery by erythrocytes: “Primum non nocere”. Transfus Apher Sci. 2016 Dec; 55(3): 275–280.). In recent years, some products that carry drugs on red blood cells have also entered clinical research. The mechanism of action of some of these products is to enter the spleen and activate its immune cells (Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15. Sivan Elloul et al. Cancer Immunol Immunother. 2021 Sep; 70(9): 2701-2719.).

[0003] Programmed cell death protein 1, also known as PD-1 or CD279, is a cell surface protein that downregulates the immune system's response to human cells by binding to its ligand, PD-L1. It regulates the immune system and promotes self-tolerance by inhibiting inflammatory T cell activity. This can prevent autoimmune diseases, but it can also prevent the immune system from killing cancer cells. PD-1 is an immune checkpoint that prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in the lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). In tumorous disease, the interaction between PD-L1 on tumor cells and PD-1 on T cells reduces T cell functional signaling, thereby preventing the immune system from attacking tumor cells. The use of inhibitors (monoclonal antibodies) that block the interaction between PD-L1 and the PD-1 receptor can reactivate some already dysfunctional cells while preventing T cells provided by the peripheral immune system from becoming tolerant T cells, thereby achieving tumor treatment. The use of anti-PD-1 monoclonal antibodies has achieved significant therapeutic effects in the clinic for cancers including melanoma, head and neck cancer, renal cell carcinoma, non-small cell lung cancer, bladder cancer, and colorectal cancer. However, among these indications, some patients still cannot benefit from PD-1 / PD-L1 antibody treatment. The main reason is that PD-1 / PD-L1 antibodies mainly act locally on tumor tissue (such as T cells), while in the patient's tumor, T cells are in a terminally exhausted state and cannot be reversed by antibody drugs, thus developing drug resistance. In the late stage of tumor, severe T cell dysfunction occurs. These T cells are difficult to be reactivated by blocking the PD1-PDL1 pathway, showing resistance to PD1 antibodies (A Burned-Out CD8+T-cell Subset Expands in the Tumor Microenvironment and Curbs Cancer Immunotherapy. Cancer Discov. 2021 Jul; 11(7): 1700-1715. doi: 10.1158 / 2159-8290.CD-20-0962. Epub 2021 Mar 3. PMID: 33658301.)(Peripheral T cell expansion predicts tumour infiltration and clinical response. Nature. 2020 Mar; 579(7798): 274-278. doi: 10.1038 / s41586-020-2056-8. Epub 2020 Feb) 26.PMID:32103181.).Therefore, the therapeutic application of PD-1 / PD-L1 antibodies faces similar problems of immune tolerance and drug resistance.

[0004] Therefore, there is still a need for more effective products for treating cancer, especially products that can better activate the peripheral immune system and / or more effectively treat cancer (e.g., cancers that cannot benefit from treatment with PD-1 or PD-L1 inhibitors).

[0005] Summary of the Invention

[0006] Existing engineering red blood cell (or other cell type) coupling technologies, used to treat specific diseases, are unable to achieve microscopic coupling of specific amino acid sites on protein drug molecules to specific proteins and sites on red blood cell (or other cell type) membrane proteins. The present invention utilizes chemical small molecule linkers, such as SMCC, to couple specific amino acid sites on protein macromolecules at one end and specific proteins and amino acid sites on red blood cells at the other end, achieving targeted protein coupling to red blood cells. This invention can enable engineered red blood cells to have better safety, DMPK, and efficacy than proteins alone.

[0007] The present invention is based on the following findings:

[0008] 1. Through mass spectrometry analysis, we discovered the amino acid sites and ratios of chemical small molecule linkers such as SMCC that connect antibodies (such as the anti-PD-1 antibody Keytruda).

[0009] 2. By optimizing the reaction conditions, the number of coupling sites can be reduced and the percentage of single positions of linkers (such as SMCC) connected to antibodies (such as the anti-PD-1 antibody Keytruda) can be increased.

[0010] 3. Through mass spectrometry analysis, it was found that specific amino acids of antibodies (such as the anti-PD-1 antibody Keytruda) can be connected to cell membrane proteins (such as red blood cells) through chemical small molecule linkers such as SMCC

[0011] 4. The K amino acid of the Fc segment of the antibody's heavy chain can be connected by a small molecule chemical linker such as SMCC, such as the Fc sequence of IgG1, IgG2, IgG3, IgG4 or its mutant sequence; further, such as the K43, K64, K81, K190 sites of the BeiGene PD1 antibody heavy chain sequence; the K199, K246, K248 sites of the Merck PD1 antibody heavy chain sequence.

[0012] 5. The K amino acid in the constant region of the light chain Kappa chain of the antibody can be connected by a small molecule linker such as SMCC, such as the PD-L1 antibodies of Roche and AZ; further, such as the K amino acid on the ADYEKHK sequence in their Kappa chain constant region.

[0013] 6. All cell membrane proteins, such as all red blood cells (such as mature red blood cells from peripheral blood and mature red blood cells differentiated from stem cells), can be treated with reducing agents, such as TCEP or its salts, such as its hydrochloride, to open the C amino acid disulfide bond, thereby reacting with linkers, such as SMCC, and then connecting proteins, such as antibodies, through linkers.

[0014] 7. Mass spectrometry analysis has identified the C amino acid positions and ratios of small molecule linkers, such as SMCC, that bind to erythrocytes (e.g., mature erythrocytes derived from peripheral blood or differentiated from stem cells). For example, they bind to the Band3 protein on the erythrocyte membrane, specifically the C amino acid 21 of the Band3 protein sequence SGDPSQPLLPQHSSLETQLFCEQGDGGTEGHSPSGILEK.

[0015] 8. The engineered red blood cells obtained by the above method can have better activity than the antibodies themselves and can be used to treat various diseases such as cancer, autoimmune, and metabolic diseases.

[0016] As a new type of drug delivery carrier, erythrocytes can improve the in vivo efficacy of the drug by changing the biological distribution of the drug in vivo, and can carry a variety of drugs. In the present invention, for example, erythrocytes carry antibodies, and through the natural biological metabolic function of erythrocytes, the antibodies carried (such as PD-1 antibodies or PD-L1 antibodies) can be distributed in the spleen tissue rich in the immune system, especially the red pulp area, which is rich in a large number of immunosuppressive cells. Erythrocytes carrying antibodies can effectively relieve the inhibitory effect of these immunosuppressive cells on peripheral T cells, thereby more effectively activating peripheral immunity. A new treatment strategy is provided for the problem of immune resistance of antibodies, such as PD-1 or PD-L1 antibodies.

[0017] The present invention therefore provides red blood cells carrying therapeutic drugs (such as immune checkpoint (such as PD-1 or PD-L1) inhibitors, or immune checkpoint activators). In some embodiments, the drug is covalently linked to the surface of the red blood cell membrane by chemical covalent coupling. In some embodiments, the red blood cells are natural adult red blood cells. The present invention successfully achieves the transformation of adult natural red blood cells through chemical modification methods, so that natural red blood cells can efficiently carry therapeutic drugs. This technology platform can significantly increase the cell drug load, reduce red blood cell damage, and achieve long-term stability in vivo and in vitro, so as to better exert the biological advantages of red blood cell drugs in the body.

[0018] In a specific embodiment, the present invention provides red blood cells loaded with a PD-1 inhibitor or a PD-L1 inhibitor, in particular red blood cells loaded with an antibody such as a PD-1 antibody or a PD-L1 antibody (red blood cell-PD1 or red blood cell-PDL1 antibody conjugate, or RBC-Anti-PD1 or Anti-PD1-RBC; or RBC-Anti-PDL1 or Anti-PDL1-RBC).

[0019] In a specific embodiment, the present invention successfully developed a cellular drug consisting of engineered red blood cells coupled to a PD-L1 antibody (αPD-L1-RBC). αPD-L1-RBC leverages the enrichment of red blood cells in the spleen, a lymphoid organ, to reshape the spleen's immune system, fully stimulating anti-tumor immune responses and thereby overcoming the problem of immune resistance to the PD-L1 antibody.

[0020] Therefore, the antibody-erythrocyte conjugates of the present invention have one or more of the following properties, in particular, have one or more of the following improved properties compared to the corresponding inhibitors not loaded on erythrocytes:

[0021] 1) Improve the efficacy of the inhibitor carried, for example, for cancers such as lung cancer, pancreatic cancer, breast cancer, gastrointestinal cancer such as colon cancer, colorectal cancer or rectal cancer, etc., with better tumor suppression effect and / or lower effective dose; specifically, for example

[0022] (a) In inhibitor-sensitive colon cancer, such as in the in vivo inhibitor-sensitive mouse MC38 tumor model, it has a better tumor inhibitory effect;

[0023] (b) A lower effective dose in inhibitor-sensitive colon cancer, such as in the in vivo inhibitor-sensitive mouse MC38 tumor model;

[0024] 2) Effective for cancers that are resistant to or insensitive to inhibitors; for example, for lung cancer, pancreatic cancer, breast cancer, gastrointestinal cancer such as colon cancer, colorectal cancer or rectal cancer that are insensitive to inhibitor treatment, it has better tumor suppression effect and / or has a lower effective dose; specifically, for example

[0025] (a) In inhibitor-insensitive lung cancer, such as in the in vivo inhibitor-insensitive mouse lung cancer KP model, it has a better tumor inhibitory effect;

[0026] (b) It has a better tumor suppressive effect in inhibitor-insensitive breast cancer, such as in the in vivo inhibitor-insensitive mouse breast cancer PB3 model;

[0027] 3) Have stronger PD1-PDL1 binding blocking ability, for example, in in vitro cell activity experiments, have stronger PD1-PDL1 binding blocking ability than the same dose of PD1 inhibitors;

[0028] 4) improving the distribution of the inhibitor in vivo (e.g., in mice or humans), for example, by enriching a higher proportion of the inhibitor in the spleen and / or activating more effector CD8+ T cells in the spleen;

[0029] 5) Activate a higher proportion of CD8+ T cells compared to the corresponding inhibitor at the same dose;

[0030] 6) Compared with the corresponding inhibitors at the same dose, it better activates the immune system and immune cells, such as activating more immunotherapy markers. For example, compared with the corresponding inhibitors not loaded on red blood cells, it can more significantly activate the NFAT reporter downstream Luciferous fluorescence signal (RFU) at a lower dose, indicating that it can better activate the immune system and immune cells;

[0031] 7) It has a certain degree of safety in acute toxicity experiments, such as acute toxicity experiments in mice;

[0032] 8) It has good drugability.

[0033] The present invention also provides a method for engineering adult natural red blood cells to enable them to be used to efficiently carry therapeutic drugs. Compared to methods for engineering red blood cells derived from stem cells, the red blood cell engineering method of the present invention offers one or more advantages, such as convenient material collection, a simple process flow, and a shorter time from peripheral blood collection to obtaining engineered red blood cells, for example, only two days.

[0034] The present invention also provides a method for covalently linking therapeutic drugs to red blood cells, for example, to red blood cell membrane proteins, to obtain engineered red blood cells carrying the therapeutic drugs. Compared to known hypotonic methods, this method causes less damage to red blood cells, for example, without disrupting the physiological structure of red blood cells.

[0035] In some embodiments, the therapeutic agent is a protein drug molecule. In some embodiments, the therapeutic agent is an antibody. In some embodiments, the therapeutic agent is an immune checkpoint inhibitor or immune checkpoint agonist, such as an antibody that targets an immune checkpoint. In some embodiments, the immune checkpoint is selected from PD-1 or PD-L1. In some embodiments, the therapeutic agent is a PD-1 inhibitor, such as a PD-1 antibody. In some embodiments, the therapeutic agent is a PD-L1 inhibitor, such as a PD-L1 antibody

[0036] The present invention also provides a method for treating diseases, such as tumors, such as cancer, using the red blood cells carrying therapeutic drugs of the present invention.

[0037] The present invention also provides the use of red blood cells carrying therapeutic drugs for treating diseases, such as tumors, such as cancer.

[0038] The present invention also provides the use of red blood cells carrying therapeutic drugs in the preparation of drugs for treating diseases, such as tumors, such as cancer. Description of the drawings:

[0039] Figure 1A shows the results of the immunohistochemical staining experiment of RBC-Keytruda, and Figures 1B and 1C show the results of the PD-1 / PD-L1 blocking bioassay. Figure 1B shows the comparison of the in vitro activity of RBC-Keytruda and Keytruda at the same low dose; Figure 1C shows the comparison of the in vitro activity of RBC-Keytruda and Keytruda at the same Keytruda dose.

[0040] Figure 2 shows the change curve of mouse tumors.

[0041] Figure 3 shows the mouse survival rate curve.

[0042] Figure 4 shows the change curve of mouse tumors.

[0043] FIG5 shows the changes in the total number of tumor-infiltrating CD8+ cells in mice.

[0044] Figure 6 shows the change curve of mouse tumors.

[0045] FIG7 shows the comparison of endpoint tumor size.

[0046] FIG8 shows the proportion of Cell Trace Far Red positive cells in mouse peripheral blood.

[0047] Figure 9 shows the Keytruda-positive ratio among Cell Trace Far Red-positive cells.

[0048] FIG10 shows an exemplary method for constructing antibody-erythrocyte conjugates.

[0049] FIG11 shows the results of coupling efficiency testing of antibody-erythrocyte conjugates.

[0050] FIG12 shows the results of in vitro activity assay of antibody-erythrocyte conjugates.

[0051] FIG13 shows the results of in vivo pharmacokinetic testing of antibody-erythrocyte conjugates.

[0052] Figure 14 shows the peptide map UV spectrum of the coupling site analysis of SMCC-Keytruda.

[0053] FIG15 shows the peptide map UV spectrum of the coupling site analysis of SMCC-Tislelizumab.

[0054] Figure 16 shows the comparative results of Keytruda processing with SMCCs of different lengths (different linkers).

[0055] FIG17 shows the mass spectrometry detection results of SMCC-GGG peptide modification and the membrane proteins modified therewith.

[0056] Figure 18 shows the peptide map UV spectrum of the SMCC-ZJM&TSQ sample.

[0057] FIG19 shows a schematic diagram of the preparation process of RBCs-αPD-L1.

[0058] FIG20 shows the evaluation of RBCs-αPD-L1 drug loading.

[0059] Figure 21 shows the RBCs-αPD-L1 drug-time curve. The engineered red blood cell preparation was administered to each group of mice on D0, D3, D6, and through the tail vein. Whole blood was collected from each group of mice 30 minutes after administration on D0, before administration on D3, 30 minutes after administration on D3, before administration on D6, 30 minutes after administration on D6, and on D7, D9, D12, and D15 for flow cytometry analysis of the proportion of engineered red blood cells. Statistical analysis was performed using GraphPad Prism 9.

[0060] Figure 22 shows the pharmacodynamic evaluation of RBCs-αPD-L1 in an αPD-L1 antibody-responsive tumor model (hPD-L1+MC38). The engineered red blood cell preparation was administered via the tail vein on D0, D3, D6, and D9, and tumors were measured on D0, D3, D7, D9, and D11. The data were statistically analyzed using GraphPad Prism 9 to generate tumor growth curves.

[0061] Figure 23 shows the pharmacodynamic evaluation of RBCs-αPD-L1 (RBCs-αPD-L1-1 and αPD-L1-3) in a tumor model (hPD-L1-MC38) that does not respond to αPD-L1 antibodies. The engineered red blood cell preparations were administered via the tail vein of each group of mice on D0, D3, D6, D9, and D11, and tumors were measured on D0, D4, D7, D11, and D14. The data were statistically analyzed using GraphPad Prism 9 to generate tumor growth curves.

[0062] Figure 24 shows the pharmacodynamic evaluation of RBCs-αPD-L1 (RBCs-αPD-L1-1 and αPD-L1-4) in a tumor model (hPD-L1-MC38) that does not respond to αPD-L1 antibodies. The engineered red blood cell preparation was administered via the tail vein of each group of mice on D0, D2, D4, D6, and D9, and tumors were measured on D0, D2, D5, D7, and D10. The data were statistically analyzed using GraphPad Prism 9 to generate tumor growth curves.

[0063] Detailed description of the invention:

[0064] I. Definition

[0065] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0066] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0067] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0068] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0069] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0070] As used herein, protein drug molecules refer to any protein that can be used as a drug, such as antibodies, such as full-length antibodies, bispecific antibodies, or multispecific antibodies; fusion proteins, such as fusion proteins comprising a receptor and an antibody fragment; and other proteins.

[0071] As used herein, the terms "anti-(human) PD-1 antibody," "anti-(human) PD-1," "(human) PD-1 antibody," "antibody that binds to (human) PD-1," or "antibody that specifically binds to (human) PD-1" refer to antibodies that bind to (human) PD-1 with sufficient affinity so that the antibody can be used as a therapeutic agent targeting (human) PD-1. In one embodiment, the (human) PD-1 antibody binds to (human) PD-1 with high affinity in vitro or in vivo. A variety of anti-PD-1 antibodies that can be used for treatment are known in the art, such as commercially available antibodies, such as Merck's pembrolizumab. Hengrui's carrelizumab, BeiGene's tislelizumab, Innovent Biologics' sintilimab, Junshi Biosciences' toripalimab, Yuheng Bio's sepalimab, Sinopharm's paimulimab, Lepu Bio's putalimab, Fuhong Hanlin's slulizumab or BMS's nivolumab.

[0072] As used herein, the terms "anti-(human) PD-L1 antibody," "anti-(human) PD-L1," "(human) PD-L1 antibody," "antibody that binds to (human) PD-L1," or "antibody that specifically binds to (human) PD-L1" refer to antibodies that can bind to (human) PD-L1 with sufficient affinity so that the antibody can be used as a therapeutic agent targeting (human) PD-L1. In one embodiment, the (human) PD-L1 antibody binds to (human) PD-L1 with high affinity in vitro or in vivo. Various anti-PD-L1 antibodies that can be used for treatment are known in the art, such as commercially available antibodies, such as atezolizumab or sugemalimab.

[0073] The terms "complete antibody", "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of a natural immunoglobulin molecule. In the case of a conventional four-chain IgG antibody, a full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy chain antibody having only heavy chains and lacking light chains, a full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds.

[0074] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0075] "Antigen-binding fragment" refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; diabodies or fragments thereof; or camelid antibodies.

[0076] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will appreciate that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. In some embodiments, the antigen is PD-1, such as human PD-1. As used herein, the term "epitope" refers to a portion of an antigen (e.g., PD-1) that specifically interacts with an antibody molecule.

[0077] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and North's CDR definitions based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Concepts", Journal of Molecular Biology, 406, 228-256 (2011)).

[0078] The following are the regional ranges of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0079] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0080] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0081] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the "Kabat numbering system" described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0082] In some embodiments, the CDRs of the antibodies of the invention are determined based on the Kabat protocol.

[0083] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Herein, the term "Fc region" does not include the heavy chain variable region VH and light chain variable region VL and the heavy chain constant region CH1 and light chain constant region CL of an immunoglobulin, but may include the hinge region at the N-terminus of the heavy chain constant region in some cases.

[0084] An "IgG-type antibody" refers to an antibody of the IgG type to which the heavy chain constant region belongs. For example, an IgG4-type antibody refers to an antibody of the IgG4-type, or an IgG1-type antibody refers to an antibody of the IgG1-type.

[0085] Red blood cells, or RBCs, are the most common type of blood cell and the primary carrier of oxygen through the bloodstream and circulatory system in vertebrates to body tissues. The cytoplasm of RBCs is rich in hemoglobin, an iron-containing biomolecule that binds oxygen and gives cells and blood their red color. The cell membrane, composed of proteins and lipids, provides properties essential for physiological cellular function, such as deformability and stability, while also allowing passage through the circulatory system, particularly the capillary network. In humans, mature RBCs are flexible, oval, biconcave discs. They lack a nucleus and most organelles to maximize the space for hemoglobin; they can be considered bags for hemoglobin, with the plasma membrane serving as a sack. Approximately 2.4 million new RBCs are produced in adults every second. RBCs make up approximately 84% of the cells in the human body, numbering 20–30 trillion. Nearly half of the blood volume (40% to 45%) is RBCs.

[0086] "Adult red blood cells" encompass both natural adult red blood cells and mature red blood cells derived from stem cells.

[0087] “Adult natural red blood cells” refer to mature natural red blood cells directly isolated from the blood (eg, peripheral blood) of an animal, particularly a human (eg, an adult or a child).

[0088] As used herein, "blood preparations" or "blood products" are used interchangeably and refer to products prepared from (human) blood for medical use. Blood preparations include whole blood preparations, blood component preparations, plasma preparations, or leukoreduced blood preparations.

[0089] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating a disease, such as a tumor, such as cancer, including chemotherapeutic agents, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants or immunoagonists).

[0090] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0091] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0092] The term "small molecule drug" refers to a low molecular weight compound that is capable of modulating biological processes. A "small molecule" is defined as a molecule having a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than larger molecules.

[0093] The term "immunomodulator" as used herein refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants or immune agonists. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0094] The term "effective amount" refers to an amount or dosage of an antibody or fragment or composition or formulation or preparation or combination or combination product of the present invention that produces the desired effect in a patient in need of treatment or prevention after single or multiple doses of the antibody or fragment or composition or formulation or preparation or combination or combination product of the present invention.

[0095] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome at the required dosage and for the required period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or formulation or product or combination or combination product are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 30%, even more preferably by at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0096] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0097] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0098] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0099] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0100] An "isolated" antibody or other molecule (e.g., an ADC molecule or nucleic acid molecule) is one that has been separated from a component of its natural environment or the environment in which it is expressed. In some embodiments, the antibody or ADC molecule is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0101] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0102] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and hematological tumors.

[0103] The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the present invention include lung cancer, pancreatic cancer, breast cancer, gastrointestinal tumors such as colon cancer, colorectal cancer or rectal cancer, etc., including metastatic forms of those cancers.

[0104] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" as used herein are not mutually exclusive.

[0105] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0106] The term "pharmaceutical composition" refers to a composition that is in form permitting the active ingredient contained therein to be effective for biological activity, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0107] The term "drug combination" refers to a non-fixed combination product or a fixed combination product, including but not limited to a medicine box, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) the protein drug molecule-erythrocyte conjugate of the present invention and (ii) other therapeutic agents) are administered to the patient simultaneously, without specific time restrictions or at the same or different time intervals, in sequence as separate entities, wherein such administration provides two or more active ingredients at effective levels of prevention or treatment in the patient's body. In some embodiments, the protein drug molecule-erythrocyte conjugate and other therapeutic agents of the present invention used in the drug combination are administered at a level no greater than when they are used alone. The term "fixed combination" means that two or more active ingredients are administered to the patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of two or more active ingredients are selected so that the combined use of each part can produce an effect greater than that achieved by using any one ingredient alone when treating a disease or condition. Each ingredient can be in the form of a separate formulation, which can be the same or different.

[0108] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). The powder and / or liquid can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent or treatment modality in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of combination therapy in treating disorders or conditions described herein.

[0109] As used herein, "treat," ...

[0110] As used herein, "prevention" includes the inhibition of the development or progression of a disease or condition, or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug before the development of signs or symptoms of cancer, particularly in a subject at risk for cancer.

[0111] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0112] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0113] II. Antibody-erythrocyte conjugates

[0114] The present disclosure relates to a protein drug molecule-erythrocyte conjugate, which comprises an erythrocyte coupled to a protein drug molecule. Preferably, the protein drug molecule is coupled to the erythrocyte via a linker, preferably the protein drug molecule is coupled to a membrane protein (e.g., band 3 protein) on the erythrocyte, for example, the protein drug molecule is coupled to a membrane protein (e.g., band 3 protein) on the erythrocyte via a linker, preferably covalently coupled.

[0115] In some embodiments, the erythrocytes are adult natural erythrocytes, preferably adult natural erythrocytes from humans. In some embodiments, the erythrocytes are mature erythrocytes differentiated from stem cells. In some embodiments, the erythrocytes express erythrocyte exchange protein 1 protein (Band3 protein) on the cell membrane. In some embodiments, the Band3 protein contains cysteine, for example, it contains the fragment SGDPSQPLLPQHSSLETQLFCEQGDGGTEGHSPSGILEK (SEQ ID NO: 21).

[0116] In one embodiment, the protein drug molecule can be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 linkers. In one embodiment, the number of protein drug molecules on a single red blood cell can be 10 3 -10 6 between, for example, about 10 4 -10 6 Between or 10 5 -10 6 between, for example, about 1×105 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 or 9×10 5 indivual.

[0117] In one embodiment, the protein drug molecule of the present invention comprises an Fc region, and / or a light chain constant region, wherein the Fc region comprises one or more lysines, and / or the light chain constant region comprises one or more lysines.

[0118] In some embodiments, the protein drug molecule of the present invention is an antibody or an antigen-binding fragment thereof. In some embodiments, the protein drug molecule is an immune checkpoint inhibitor or immune checkpoint agonist, such as an antibody targeting an immune checkpoint. In some embodiments, the immune checkpoint is selected from PD-1 or PD-L1. In some embodiments, the therapeutic drug is a PD-1 inhibitor, such as a PD-1 antibody. In some embodiments, the therapeutic drug is a PD-L1 inhibitor, such as a PD-L1 antibody.

[0119] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are antibodies or antigen-binding fragments thereof that bind to PD-1, in particular human PD-1. In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are antibodies or antigen-binding fragments thereof that bind to PD-L1, in particular human PD-L1.

[0120] Any PD-1 antibody or antigen-binding fragment thereof known to those skilled in the art is suitable for use in the present invention, including but not limited to disclosed antibodies or commercially available antibodies, such as those disclosed in the following patents or patent applications: CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1 or WO2006121168A1, or pembrolizumab (Pembrolizumab). rolizumab), Hengrui's Camrelizumab, BeiGene's Tislelizumab, Innovent Biologics' Sintilimab, Junshi Biosciences' Toripalimab, Yuheng Bio's Sepalimab, Zhengda Tianqing's Painpulimab, Lepu Bio's Putelimab, Fuhong Hanlin's Slulizumab or BMS's Nivolumab. Any PD-L1 antibody or antigen-binding fragment thereof known to those skilled in the art is suitable for the present invention, including but not limited to already disclosed antibodies or commercially available antibodies, such as antibodies disclosed in the following patents or patent applications: CN102245640A or CN106432501A or CN107849130A or CN102918058A, or Envafolimab or Sugemalimab or Envafolimab or Durvalumab.

[0121] In some embodiments, antibodies suitable for use in the present invention are antibodies in the form of IgG1, antibodies in the form of IgG2, antibodies in the form of IgG3, or antibodies in the form of IgG4.

[0122] In some embodiments, antibodies suitable for use in the present invention are monoclonal antibodies. In some embodiments, antibodies suitable for use in the present invention are humanized. In some embodiments, antibodies suitable for use in the present invention are human antibodies. In some embodiments, antibodies suitable for use in the present invention are chimeric antibodies.

[0123] In one embodiment, antibodies suitable for use in the present invention are full-length antibodies.

[0124] In one embodiment, the antigen-binding fragment of the antibody suitable for use in the present invention is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), bivalent antibody or fragment thereof, linear antibody or half antibody. In some embodiments, the antigen-binding fragment suitable for use in the present invention includes but is not limited to VHH, Fv molecule, scFv molecule, Fab molecule, and F(ab')2 molecule.

[0125] In one embodiment, an antibody or antigen-binding fragment thereof suitable for use in the present invention comprises an Fc region, preferably the Fc region comprises lysine or is engineered to comprise lysine. In one embodiment, an antibody or antigen-binding fragment thereof suitable for use in the present invention comprises a light chain constant region, for example, the light chain constant region comprises lysine or is engineered to comprise lysine. In one embodiment, an antibody or antigen-binding fragment thereof suitable for use in the present invention comprises an Fc region and a light chain constant region, preferably the Fc region comprises lysine or is engineered to comprise lysine and the light chain constant region comprises lysine.

[0126] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof against PD-1 is an anti-PD-1 antibody or antigen-binding fragment thereof disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1 or WO2006121168A1. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises CN108473977B, CN104250302B, One or more CDRs (preferably three CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably six CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of an anti-PD-1 antibody or antigen-binding fragment thereof disclosed in CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or VH and / or VL comprising an anti-PD-1 antibody or antigen-binding fragment thereof disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or the heavy chain and / or light chain comprising said antibody.

[0127] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof against PD-1 is pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulizumab or nivolumab or an antigen-binding fragment thereof. In one embodiment, the anti-PD-1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penampalimab, putalimab, slulizumab, or nivolumab, or an antigen-binding fragment thereof, or comprises the VH and / or VL of pembrolizumab, carrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penampalimab, putalimab, slulizumab, or nivolumab, or an antigen-binding fragment thereof, or comprises the heavy chain and / or light chain of said antibody.

[0128] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from the heavy chain variable region and three complementarity determining regions (LCDRs) from the light chain variable region.

[0129] In some aspects, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH). In some aspects, the anti-PD-1 antibody or its antigen-binding fragment comprises a light chain variable region (VL). In some aspects, the anti-PD-1 antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0130] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof comprises an antibody heavy chain. In some embodiments, the anti-PD-1 antibody heavy chain comprises a heavy chain variable region and a heavy chain constant region. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof suitable for use in the present invention comprises an antibody light chain. In some embodiments, the anti-PD-1 antibody light chain of the present invention comprises a light chain variable region and a light chain constant region. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof suitable for use in the present invention comprises a heavy chain and a light chain.

[0131] In some embodiments, the heavy chain variable region is any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or the heavy chain variable region of pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulimab, or nivolumab. In some embodiments, the light chain variable region is the light chain variable region of any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulimab, or nivolumab. In some embodiments, the heavy chain variable region CDR1, CDR2, or CDR3 is any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or the heavy chain variable region CDR1, CDR2, or CDR3 of pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penampalimab, putalimab, slulimab, or nivolumab. In some embodiments, the light chain variable region CDR1, CDR2, or CDR3 is the light chain variable region CDR1, CDR2, or CDR3 of any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulimab, or nivolumab. In some embodiments, the heavy chain is any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or the heavy chain of pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulimab, or nivolumab.In some embodiments, the light chain is the light chain of any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulimab, or nivolumab.

[0132] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2 and CDR3, and the light chain variable region CDR1, CDR2 and CDR3 from any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1 or WO2006121168A1, or pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penampalimab, putalimab, slulizumab or nivolumab.

[0133] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from any antibody disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penpulimab, putalimab, slulimab, or nivolumab.

[0134] In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present invention comprise the heavy and light chains of any of the antibodies disclosed in CN108473977B, CN104250302B, CN105531288B, CN105026428B, WO2008156712A1, or WO2006121168A1, or pembrolizumab, camrelizumab, tislelizumab, toripalimab, sintilimab, sepalimab, penampalimab, putalimab, slulizumab, or nivolumab. In some embodiments, the anti-PD-1 antibodies or antigen-binding fragments thereof of the present invention comprise the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3, of pembrolizumab.

[0135] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from pembrolizumab.

[0136] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from pembrolizumab.

[0137] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from camrelizumab.

[0138] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from camrelizumab.

[0139] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from camrelizumab.

[0140] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from tislelizumab.

[0141] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from tislelizumab.

[0142] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from tislelizumab.

[0143] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from sintilimab.

[0144] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from sintilimab.

[0145] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from sintilimab.

[0146] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from toripalimab.

[0147] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from toripalimab.

[0148] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from toripalimab.

[0149] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from sepalimumab.

[0150] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from sepalimumab.

[0151] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from sepalimumab.

[0152] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from penpulimab.

[0153] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from penpulimab.

[0154] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from penpulimab.

[0155] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from putalimab.

[0156] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from putalimab.

[0157] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from putalimab.

[0158] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from slulizumab.

[0159] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from slulizumab.

[0160] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from slulizumab.

[0161] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from nivolumab.

[0162] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region from nivolumab.

[0163] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from nivolumab.

[0164] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof against PD-L1 is an anti-PD-L1 antibody or antigen-binding fragment thereof disclosed in CN102245640A or CN106432501A or CN102918058A. In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably three CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably six CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of an anti-PD-L1 antibody or antigen-binding fragment thereof disclosed in CN102245640A or CN106432501A or CN102918058A, or comprises VH and / or VL of an anti-PD-L1 antibody or antigen-binding fragment thereof disclosed in CN102245640A or CN106432501A or CN102918058A, or comprises the heavy chain and / or light chain of said antibody.

[0165] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably three CDRs, i.e., HCDR1, HCDR2H, and HCDR3) of the anti-PD-L1 antibody or antigen-binding fragment thereof disclosed in CN107849130A, or comprises the VHH of the anti-PD-L1 antibody or antigen-binding fragment thereof disclosed in CN107849130A, or comprises the heavy chain of the antibody or a fusion antibody of the VHH and Fc.

[0166] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof against PD-L1 is sugemalimab, atezolizumab, durvalumab, or envoralizumab, or an antigen-binding fragment thereof.

[0167] In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H, and HCDR3; or LCDR1, LCDR2, and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of sugemalimab, atezolizumab, or durvalumab, or an antigen-binding fragment thereof, or comprises the VH and / or VL of sugemalimab, atezolizumab, or durvalumab, or an antigen-binding fragment thereof, or comprises the heavy chain and / or light chain of said antibody. In one embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H, and HCDR3) of envomalimab, or an antigen-binding fragment thereof, or comprises the VHH of envomalimab, or an antigen-binding fragment thereof, or comprises a fusion antibody comprising the VHH of said antibody and an Fc region.

[0168] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from a heavy chain variable region or a single domain antibody (VHH), namely, HCDR1, HCDR2, and HCDR3. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (LCDRs) from a light chain variable region, namely, LCDR1, LCDR2, and LCDR3. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0169] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) or a single-domain antibody VHH. In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a light chain variable region (VL). In some aspects, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the VHH comprises three complementary determining regions (CDRs) from VHH, HCDR1, HCDR2, and HCDR3.

[0170] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an antibody heavy chain. In some embodiments, the anti-PD-L1 antibody heavy chain comprises a heavy chain variable region and a heavy chain constant region. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof suitable for use in the present invention comprises an antibody light chain. In some embodiments, the anti-PD-L1 antibody light chain of the present invention comprises a light chain variable region and a light chain constant region. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof suitable for use in the present invention comprises a heavy chain and a light chain.

[0171] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a single-domain antibody VHH and an Fc region, such as a VHH fused to an Fc region.

[0172] In some embodiments, the heavy chain variable region is any antibody disclosed in CN102245640A or CN106432501A or CN102918058A, or the heavy chain variable region of sugemalimab or atezolizumab or durvalumab. In some embodiments, the VHH is any antibody disclosed in CN107849130A or the VHH of envoralizumab. In some embodiments, the light chain variable region is any antibody disclosed in CN102245640A or CN106432501A or CN102918058A, or the light chain variable region of sugemalimab or atezolizumab or durvalumab. In some embodiments, the heavy chain variable region CDR1, CDR2, or CDR3 is any antibody disclosed in CN102245640A, CN106432501A, or CN102918058A, or the heavy chain variable region CDR1, CDR2, or CDR3 of sugemalimab, atezolizumab, or durvalumab. In some embodiments, the heavy chain variable region or VHH CDR1, CDR2, or CDR3 is any antibody disclosed in CN107849130A, or the VHH CDR1, CDR2, or CDR3 of envolimab. In some embodiments, the light chain variable region CDR1, CDR2 or CDR3 is any antibody disclosed in CN102245640A or CN106432501A or CN102918058A, or the light chain variable region CDR1, CDR2 or CDR3 of sugemalimab or atezolizumab or durvalumab. In some embodiments, the heavy chain is any antibody disclosed in CN102245640A or CN106432501A or CN102918058A, or the heavy chain of sugemalimab or atezolizumab or durvalumab. In some embodiments, the VHH fused to the Fc region is any VHH fused to the Fc region disclosed in CN107849130A or envoralizumab. In some embodiments, the light chain is the light chain of any antibody disclosed in CN102245640A or CN106432501A or CN102918058A or sugemalimab or atezolizumab or durvalumab.

[0173] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from any antibody disclosed in CN102245640A, CN106432501A, or CN102918058A, or sugemalimab, atezolizumab, or durvalumab.

[0174] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises CDR1, CDR2, and CDR3 from any antibody disclosed in CN107849130A or the VHH of Envolimab.

[0175] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from any antibody disclosed in CN102245640A or CN106432501A or CN102918058A, or sugemalimab or atezolizumab or durvalumab.

[0176] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a VHH from any of the antibodies disclosed in CN107849130A or Envolimab.

[0177] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain and light chain from any antibody disclosed in CN102245640A or CN106432501A or CN102918058A or sugemalimab or atezolizumab or durvalumab.

[0178] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises any VHH and Fc fusion antibody disclosed in CN107849130A or Envolimab.

[0179] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from atezolizumab.

[0180] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from atezolizumab.

[0181] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from atezolizumab.

[0182] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from sugemalimab.

[0183] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from sugemalimab.

[0184] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from sugemalimab.

[0185] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises VHH CDR1, CDR2, and CDR3 from Envolimab.

[0186] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a VHH from Envolimab.

[0187] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises envoralizumab.

[0188] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region CDR1, CDR2, and CDR3, and the light chain variable region CDR1, CDR2, and CDR3 from durvalumab.

[0189] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises the heavy chain variable region and the light chain variable region from durvalumab.

[0190] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain from durvalumab.

[0191] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises heavy chain variable regions CDR1 (HCDR1), CDR2 (HCDR2) and CDR3 (HCDR3), and light chain variable regions CDR1 (LCDR1), CDR2 (LCDR2) and CDR3 (LCDR3), wherein

[0192] (i) the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10; and the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 3, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 4, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5;

[0193] (ii) the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 18, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 19, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20; and the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15; or

[0194] (iii) the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:30, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:31, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:32; and the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:25, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:26, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:27.

[0195] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0196] (i) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7, or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said amino acid sequence, and

[0197] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 2, or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence;

[0198] (ii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 17, or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said amino acid sequence, and

[0199] The light chain variable region comprises the amino acid sequence of SEQ ID NO: 12, or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence; or

[0200] (iii) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 29, or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said amino acid sequence, and

[0201] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO:24, or has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0202] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0203] (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or consists of the amino acid sequence, and

[0204] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the amino acid sequence;

[0205] (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, and

[0206] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12, or consists of the amino acid sequence; or

[0207] (iii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, and

[0208] The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, or consists of the amino acid sequence.

[0209] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0210] (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 6, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said amino acid sequence, and

[0211] The light chain comprises the amino acid sequence of SEQ ID NO: 1, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence;

[0212] (ii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 16, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said amino acid sequence, and

[0213] The light chain comprises the amino acid sequence of SEQ ID NO: 1, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence; or

[0214] (iii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 28, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to said amino acid sequence, or consists of said amino acid sequence, and

[0215] The light chain comprises the amino acid sequence shown in SEQ ID NO:23, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0216] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain and a light chain, wherein

[0217] (i) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the amino acid sequence, and

[0218] The light chain comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the amino acid sequence;

[0219] (ii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, and

[0220] The light chain comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the amino acid sequence; or

[0221] (iii) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 28, and

[0222] The light chain comprises the amino acid sequence shown in SEQ ID NO: 23, or consists of the amino acid sequence.

[0223] In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the present invention comprises a fusion antibody of VHH and Fc, which comprises the amino acid sequence shown in SEQ ID NO: 22, or has an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence, or consists of the amino acid sequence.

[0224] In some embodiments, a linker can be used to connect one or more protein drug molecules, such as an antibody or its antigen-binding fragment, to an erythrocyte (e.g., a membrane protein of an erythrocyte) to form a protein drug molecule-erythrocyte conjugate, such as an antibody-erythrocyte conjugate. In some embodiments, the linker is a bifunctional linker (divalent linker). In some embodiments, a protein drug molecule-erythrocyte conjugate, such as an antibody-erythrocyte conjugate, can be prepared using a linker with a reactive functional group, which is used to covalently connect to an erythrocyte and a protein drug molecule, such as an antibody. In some embodiments, the linker reacts with a nucleophilic group of a protein drug molecule, such as an antibody or its antigen-binding fragment, and with a nucleophilic group of an erythrocyte membrane protein to form a covalent bond. For example, in some embodiments, the thiol group of a cysteine ​​residue on an erythrocyte membrane protein can form a chemical bond with a reactive functional group of a linker or a protein drug molecule-linker intermediate, such as an antibody-linker intermediate, to prepare a protein drug molecule-erythrocyte conjugate, such as an antibody-erythrocyte conjugate.

[0225] Nucleophilic groups on protein drug molecules, such as antibodies or erythrocyte membrane proteins, include, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups (in the case of glycosylated antibodies). Amino, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents including the following to form covalent bonds: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups.

[0226] In some embodiments, the nucleophilic group on a protein drug molecule, such as an antibody, is an N-terminal amino group or a side chain amino group (such as an amino group of lysine), and / or the nucleophilic group on an erythrocyte membrane protein is a side chain thiol group, such as a thiol group after reduction of cysteine.

[0227] In some embodiments, the red blood cell membrane protein is Band3.

[0228] In one embodiment, the linker has a functional group that can react with a thiol group on a cysteine ​​residue present on an RBC membrane protein to form a covalent bond to the RBC. Non-limiting exemplary reactive functional groups of this type include maleimide, haloacetamide, α-haloacetyl, active esters such as succinimidyl ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acyl chloride, sulfonyl chloride, isocyanate and isothiocyanate, preferably maleimide (MAL).

[0229] In some embodiments, the functional group of the linker can be connected to the antibody by reacting with the amino group (-NH2) on the lysine in the antibody structure. Non-limiting exemplary reactive functional groups of this type include but are not limited to active esters, such as NHS esters, HOBt esters, haloformates and acid halides, preferably N-hydroxysuccinimide esters (NHS esters).

[0230] The linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), p-aminobenzyloxycarbonyl ("PAB"), NHS esters such as N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("MCC"). The linker may also comprise an amino acid, such as one or more glycine. Various linker components are known in the art.

[0231] In some embodiments, the linker can be attached to the red blood cells RBC by reacting with the thiol group of the free cysteine ​​residue of the red blood cell membrane protein, and attached to the antibody Ab by reacting with the amino group (-NH2) on the lysine in the antibody structure.

[0232] Exemplary linkers include, but are not limited to:

[0233] Bis-maleimido-trioxyethylene glycol (BMPEO), N-(β-maleimidopropoxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy) succinimide ester (EMCS), N-[γ-maleimidobutyryloxy] succinimide ester (GMBS), 1,6-hexane-bis-vinyl sulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-aminohexanoate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 3-(bromoacetamido)propionic acid succinimidyl ester (SB AP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl-4-(2-pyridylthio) pentanoate (SPP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), NHS-PEGn-MAL (wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more, such as NHS-PE G2-MAL, NHS-PEG3-MAL, NHS-PEG6-MAL, NHS-PEG8-MAL, NHS-PEG24-MAL), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), succinimidyl 6-[(β-maleimidopropionamido) hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB, and succinimidyl (4-vinylsulfone) benzoate (SVSB), and bis-maleimide reagents such as dithiobismaleimidoethane (DTME), 1,4-dimethoxy- ... -bismaleimidobutane (BMB), 1,4-bismaleimido-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG)2 and BM(PEG)3), difunctional derivatives of imino esters (such as dimethyl diimidoadipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).In some embodiments, bismaleimide reagents allow the attachment of thiol groups of cysteine ​​residues in antibodies to thiol-containing drug moieties, linkers, or linker-drug intermediates. Other functional groups that react with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0234] In some embodiments, the linker is SMCC, such as Sulfo-SMCC, or SMCC-GGG, or NHS-PEGn-MAL NHS-PEGn-MAL (wherein n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more), such as NHS-PEG2-MAL or NHS-PEG 24 -MAL.

[0235] In some embodiments, the RBCs of the present invention are natural adult red blood cells. In some embodiments, the RBCs are mature red blood cells isolated from human (eg, adult or child) blood, or are mature red blood cells differentiated from stem cells.

[0236] In some embodiments, the RBCs are derived from mature red blood cells having the same blood type as the subject to be administered. In some embodiments, the RBCs of the present invention are capable of expressing Band3 protein or a fragment thereof. In some embodiments, the Band3 protein or its fragment comprises cysteine ​​or is engineered to comprise cysteine. In some embodiments, the Band3 protein or its fragment comprises the fragment SGDPSQPLLPQHSSLETQLFCEQGDGGTEGHSPSGILEK (SEQ ID NO: 21). In some embodiments, the red blood cells are linked to a linker (e.g., a linker comprising maleimide, e.g., linked by maleimide reaction) via a cysteine ​​on the Band3 protein or its fragment, such as cysteine ​​at position 21 of the fragment SGDPSQPLLPQHSSLETQLFCEQGDGGTEGHSPSGILEK (SEQ ID NO: 21).

[0237] In some embodiments, the RBCs are mature red blood cells (e.g., natural adult red blood cells) obtained by treatment with a thiol reducing agent, which contain cysteine ​​residues containing thiol groups. In some embodiments, the thiol reducing agent is TCEP or a salt thereof, such as its hydrochloride, tris-(2-carboxyethyl)phosphine hydrochloride.

[0238] "TCEP" is the abbreviation of "Tris(2-carboxyethyl)phosphine", and its chemical formula is C6H 16 O6P2 is commonly used as a reducing agent. In biochemistry and molecular biology, TCEP is particularly used to reduce disulfide bonds in proteins and peptides, and in protein engineering to remove sulfhydryl modifications from proteins.

[0239] In some embodiments, RBCs are obtained by a method comprising:

[0240] (i) separating and concentrating red blood cells from (human) whole blood, optionally by filtering out leukocytes;

[0241] (ii) treating erythrocytes with a thiol reducing agent (e.g., TCEP or a salt thereof, such as its hydrochloride) to chemically modify the surface of the erythrocytes;

[0242] (iii) The modified red blood cells are collected and concentrated.

[0243] In some embodiments, the surface chemical modification in (ii) comprises the following steps:

[0244] The thiol reducing agent is mixed with the concentrated red blood cells, wherein the concentration of the reducing agent is between 0.1 mM and 50 mM, such as 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 5.0 mM.

[0245] III. Methods for preparing drug protein molecule-erythrocyte conjugates (eg, antibody-erythrocyte conjugates)

[0246] The present disclosure also provides a method for preparing an antibody-erythrocyte conjugate, comprising:

[0247] (1) reacting a nucleophilic group of the antibody or antigen-binding fragment thereof with a linker (e.g., a bifunctional linker) to form an antibody or antigen-binding fragment thereof connected to the linker via a covalent bond,

[0248] (2) chemically modifying the surface of adult natural red blood cells to expose nucleophilic groups on red blood cell membrane proteins;

[0249] (3) mixing the antibody or antigen-binding fragment thereof connected to the linker in (1) and the red blood cells obtained in (2) so that the two are covalently coupled via the linker;

[0250] (4) Collect the antibody-erythrocyte conjugate obtained in (3).

[0251] Nucleophilic groups on antibodies or erythrocyte membrane proteins include, but are not limited to: (i) N-terminal amino groups, (ii) side chain amino groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups (in the case of glycosylated antibodies). Amino, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents including the following to form covalent bonds: (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups.

[0252] In some embodiments, the nucleophilic group on the antibody is an N-terminal amino group or a side chain amino group (such as the amino group of lysine), and the nucleophilic group on the red blood cell membrane protein is a side chain thiol group, such as the thiol group after reduction of cysteine.

[0253] In some embodiments, the method for preparing the antibody-erythrocyte conjugate of the present invention is shown in FIG10 or 19 , wherein linker represents a linker, such as SMCC (e.g., sulfo-SMCC), and anti-PD-1 refers to an anti-PD-1 antibody, and the method comprises:

[0254] (1) connecting the amino group on the lysine of the PD-1 or PD-L1 antibody to a linker;

[0255] (2) chemically modifying erythrocytes to expose the thiol groups of cysteine ​​residues on erythrocyte membrane proteins;

[0256] (3) The PD-1 or PD-L1 antibody obtained in (1) and connected to a linker is coupled to a cysteine ​​on an erythrocyte membrane protein via a linker.

[0257] In some embodiments, the final concentration of the linker of the invention (e.g., SMCC or sulfo-SMCC) can be in the range of 0.01-5 mM, for example, a final concentration of about 0.02 mM, 0.03 mM, 0.03125 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 In some embodiments, the final concentration of Sulfo-SMCC is about 0.03 mM, 0.25 mM, or 3 mM.

[0258] In some embodiments, the final concentration of the linker of the invention: antibody molar ratio can be 1000: 1. In some embodiments, in the method of preparing an antibody-erythrocyte conjugate, the final concentration of the linker of the invention (e.g., SMCC or Sulfo-SMCC): antibody molar ratio can be about 500: 1 or less, 400: 1 or less, 300: 1 or less, 200: 1 or less, 150: 1 or less, 130: 1 or less, 110: 1 or less, 100: 1 or less, 90: 1 or less, 50: 1 or less, 30: 1; 20: 1 or less, 10: 1 or less, 9: 1 or less, 8: 1 or less, 7: 1 or less, 6: 1 or less, 5: 1 or less, 4: 1 or less, 3: 1 or less, 2: 1 or less, 1: 1 or less, 1:5 or less, 1: 10 or less, 1:50 or less, or 1: 100 or less. Preferably, in the method of preparing the antibody-erythrocyte conjugate, the final concentration of SMCC or Sulfo-SMCC of the present invention: antibody molar ratio can be about 100: 1, 8: 1, 8.3: 1, or 1: 1. In some embodiments, the final concentration of Sulfo-SMCC: tislelizumab molar ratio can be about 100: 1, 8: 1, 8.3: 1, or 1: 1.

[0259] In some embodiments of the method for preparing an antibody-erythrocyte conjugate, the number of coupling sites can be reduced by optimizing the reaction conditions, and the percentage of a single position of the linker (such as SMCC) connected to the antibody (such as the anti-PD-1 antibody Keytruda) can be increased. In some embodiments of the method for preparing an antibody-erythrocyte conjugate, the coupling site or number on the antibody can be changed by adjusting the final concentration of SMCC, sulfo-SMCC, or the molar ratio of linker: antibody. In some embodiments of the method for preparing an antibody-erythrocyte conjugate, the percentage of a single position of SMCC or sulfo-SMCC connected to the antibody (such as tislelizumab, Keytruda) can be increased by adjusting the final concentration of SMCC or sulfo-SMCC. In some embodiments of the method for preparing an antibody-erythrocyte conjugate, the distribution and / or number of sites of lysine residues coupled to the antibody can be changed by adjusting the final concentration of sulfo-SMCC or the molar ratio of sulfo-SMCC: antibody (such as tislelizumab, Keytruda). In some embodiments of the method for preparing an antibody-erythrocyte conjugate, when the final concentration of Sulfo-SMCC is adjusted to about 31.25 uM or the molar ratio of Sulfo-SMCC to Tislelizumab is about 1.04: 1, the site to which Tislelizumab is conjugated may include K338 on the heavy chain, or substantially K338 on the heavy chain, or only K338 on the heavy chain. In some embodiments of the method for preparing an antibody-erythrocyte conjugate, when the final concentration of Sulfo-SMCC is adjusted to about 0.25 mM or the molar ratio of Sulfo-SMCC to Tislelizumab is about 8.33: 1, the site to which Tislelizumab is conjugated may include K188 on the light chain, K197 and K244 on the heavy chain; or substantially K188 on the light chain, K197 and K244 on the heavy chain; or only K188 on the light chain, K197 and K244 on the heavy chain.

[0260] In some embodiments, the method for preparing the antibody-erythrocyte conjugate of the present invention comprises:

[0261] (1) reacting a nucleophilic group of the antibody with a linker (e.g., a bifunctional linker) to form a linker connected to the antibody via a covalent bond, for example, the bifunctional linker reagent is sulfo-SMCC, preferably, the nucleophilic group is -NH2 of the lysine side chain,

[0262] Wherein said step (1) comprises,

[0263] The bifunctional linker and the antibody solution are mixed evenly, wherein the final concentration of the bifunctional linker, such as sulfo-SMCC, in the mixture is in the range of 0.01-5 mM, for example, the final concentration is about 0.02 mM, 0.03 mM, 0.03125 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.15 mM, 0.2 mM, 0.21 mM, 0.22 mM, 0.23 mM, 0.24 mM, 0.25 mM, 0.26 mM, 0.27 mM, 0.28 mM, 0.29 mM, 0.3 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, 3 mM, 3.1 mM, 3.2 mM, 3.3 mM, 3.4 mM, 3.5 mM, 4 mM or 4.5 mM, preferably a final concentration of about 0.03 mM, 0.25 mM or 3 mM;

[0264] For example, the purity of the antibody before mixing is between 10% and 100%;

[0265] For example, the final concentration of the antibody in the mixture is less than 0.33 mM;

[0266] Preferably, the mixture is mixed, for example, at a temperature in the range of 25-35 degrees Celsius (e.g., about 25-about 30° C., such as about 30° C.), for 0.5-2 hours (e.g., about 1 hour), for example, on a rotary mixer at a speed in the range of 5-15 rpm (e.g., 10 rpm);

[0267] The mixed solution is ultrafiltered, for example, 1 to 10 times.

[0268] Wherein, preferably, the buffer exchange multiple after ultrafiltration is greater than 500 times, preferably greater than 1000 times;

[0269] wherein the antibody concentration after ultrafiltration is approximately ≥5 mg / mL, ≥6 mg / mL, ≥7 mg / mL, ≥8 mg / mL, ≥9 mg / mL, ≥10 mg / mL, ≥15 mg / mL, ≥20 mg / mL, ≥25 mg / mL, ≥30 mg / mL, ≥35 mg / mL, for example, ≥10 mg / mL;

[0270] Preferably, the step (1) comprises:

[0271] Take 1mg / ml sulfo-SMCC solution, pipette 21.8μL into 138.2μL PBS buffer, and gently pipette to mix thoroughly. Pipette 40μL of anti-PD-1 antibody solution into the EP tube and mix thoroughly to a final sulfo-SMCC concentration of 0.25mM. Place the EP tube on a rotary mixer at 10rpm and 30°C for 1 hour to obtain the treated anti-PD-1 antibody protein solution.

[0272] After the reaction is completed, the treated anti-PD-1 antibody protein solution is added to the inner tube of the ultrafiltration tube, PBS buffer is added to the top white line of the ultrafiltration tube, and centrifugation is performed. After centrifugation, the upper ultrafiltration tube is removed, the waste liquid in the lower tube is poured out, the upper ultrafiltration tube is replaced, PBS buffer is added to the top white line of the ultrafiltration tube, mixed, and centrifuged. In some embodiments, ultrafiltration is performed 1-10 times;

[0273] Centrifuge until the upper layer volume is less than or equal to 100 μL (theoretical concentration of anti-PD-1 antibody protein solution ≥10 mg / mL, ≥15 mg / mL, ≥20 mg / mL, ≥25 mg / mL, ≥30 mg / mL or ≥35 mg / mL);

[0274] After centrifugation, the upper layer of anti-PD-1 antibody protein solution was aspirated and PBS was added to 100 μL (so that the theoretical concentration of the anti-PD-1 antibody protein solution was 10 mg / mL). Centrifugation parameters were: centrifugal force 3800 g, speed increase 8, speed decrease 8, temperature 4°C.

[0275] (2) chemically modifying the surface of adult natural red blood cells to expose nucleophilic groups on red blood cell membrane proteins. Preferably, the red blood cells are chemically modified using a thiol reducing agent (e.g., TCEP or a salt thereof such as its hydrochloride). Preferably, the nucleophilic group is a thiol group on cysteine.

[0276] Wherein, the step (2) includes

[0277] Separating (e.g., by filtering out leukocytes) and concentrating red blood cells from whole blood,

[0278] The thiol reducing agent is mixed evenly with the red blood cells, wherein the concentration of the reducing agent is between 0-500mM, such as 0.1mM-50mM, such as 0.5mM-10.0mM, 0.5mM-5.0mM; preferably about 5.0mM,

[0279] Preferably, the mixture is mixed, for example, at a temperature in the range of 4-37 degrees Celsius, for example, 25-35 degrees Celsius (e.g., about 25°C to about 30°C, for example, about 30°C), for a mixing time of 1 minute to 24 hours, for example, 0.5-2 hours (e.g., about 1 hour), for example, on a rotary mixer at a speed in the range of 5-15 rpm (e.g., 10 rpm);

[0280] The supernatant is removed by centrifugation. Preferably, the centrifugal force is 500-1200 g (e.g., about 800 g), the centrifugation time is 2-10 min (e.g., about 3 min), the ascending speed is 1-9 (e.g., about 9) and / or the descending speed is 1-7 (e.g., about 7);

[0281] optionally repeated twice, for example at about 20°C-30°C, for example at about 25°C;

[0282] Optionally, the modified packed red blood cells are collected.

[0283] Preferably, step (2) comprises:

[0284] Collecting a human blood sample, for example, using a commonly used clinical blood collection device or process, such as a blood collection bag; in some embodiments, the collection comprises the following steps: opening the blood collection tube cap, using a disposable syringe, transferring the human blood sample from a healthy subject in the blood collection tube to a blood bag, and then sealing the tube twice with a sterile tube sealer. Leukocytes are removed from the whole blood using a leukocyte filter, and a 20 mL disposable syringe is used to transfer the leukocyte-removed blood sample from the blood storage bag to a 50 mL centrifuge tube, followed by centrifugation to collect red blood cells, wherein the centrifugation parameters are set as follows: centrifugal force 500 g, centrifugation time 5 minutes, ramp rate 9, ramp rate 8, and temperature 25°C;

[0285] The concentrated red blood cell intermediate was obtained by centrifugation. The centrifugation parameters were set as centrifugal force 500g, centrifugation time 3 minutes, ascending speed 9, descending speed 7, and temperature 25°C.

[0286] Open the centrifuge tube, use a disposable sterile pipette or a 1000 μL pipette and matching tip to discard the supernatant liquid, use an electric pipette to aspirate the concentrated red blood cells, and add them to the reaction vessel;

[0287] Pipette 10 μL of 0.1 M TCEP or its salt, such as its hydrochloride, into the EP tube containing 90 μL of PBS buffer and mix well;

[0288] 100 μL of the concentrated red blood cells obtained above was added to the above solution and mixed evenly to make the final concentration of TCEP or a salt thereof, such as its hydrochloride, be 5.0 mM;

[0289] Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 h;

[0290] After the reaction, the supernatant was removed by centrifugation, and 5 times the volume of concentrated red blood cells of PBS buffer was added, mixed and centrifuged, and the supernatant was removed; this was repeated twice, with the centrifugation parameters being 800 g for 3 minutes, an increase speed of 9, a decrease speed of 7, and a temperature of 25°C.

[0291] Collect the modified packed red blood cells.

[0292] (3) mixing the antibody connected to the linker in (1) and the red blood cells obtained in (2) so that the two are covalently coupled via the linker;

[0293] Wherein said step (3) comprises:

[0294] Mixing the linker-linked antibody in (1) and the red blood cells obtained in (2), preferably,

[0295] The final concentration of the antibody in the mixture is 0-100 mg / ml.

[0296] Mixing the mixture, for example, at a temperature in the range of 25-35 degrees Celsius (e.g., about 25-about 37° C., such as about 30° C. or about 37° C.), for a mixing time of, for example, 1 minute to 24 hours, such as 0.5-2 hours (e.g., about 1 hour), for example, on a rotary mixer at a speed in the range of 5-15 rpm (e.g., 10 rpm);

[0297] Preferably, step (3) comprises:

[0298] Add 100 μL of the modified concentrated RBC obtained in step (2) to 100 μL of the ultrafiltered anti-PD-1 antibody protein solution obtained in step (1), and mix thoroughly by gently pipetting;

[0299] Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 h;

[0300] (4) Collect the antibody-erythrocyte conjugate obtained in (3).

[0301] The mixture obtained in step (3) is centrifuged and the supernatant removed, optionally repeated twice, for example at 20-30 degrees Celsius, for example at about 25°C.

[0302] Preferably, step (4) comprises:

[0303] The mixture obtained in step (3) was centrifuged to remove the supernatant, and 9 times the volume of physiological saline (concentrated RBC volume) was added, mixed, and centrifuged to remove the supernatant. This was repeated twice, wherein the centrifugation parameters were: centrifugal force 800 g, centrifugation time 3 minutes, ramp rate 9, ramp rate 7, temperature 25°C. The engineered red blood cells were collected.

[0304] Optionally, the method further comprises step (5): preserving the engineered red blood cells obtained in step (4),

[0305] Preferably, the step (5) includes

[0306] Pipette 100 μL of saline and 50 μL of red blood cell preservation solution (saline equal to the volume of concentrated red blood cells and 0.5 times the volume of red blood cell preservation solution (e.g., CPDA-1 red blood cell preservation solution or MAP red blood cell preservation solution)), add to the engineered red blood cells, and gently pipette to mix thoroughly to obtain the engineered red blood cell preparation solution. Add the engineered red blood cell preparation solution to the packaging container. After packaging, store at 4°C until ready to use.

[0307] In some embodiments, in the centrifugation step, the centrifugal force is 500-1200 g, the centrifugation time is 2-10 min, the rising speed is 1-9 and / or the falling speed is 1-7.

[0308] Therefore, in one embodiment, the present invention also relates to an antibody-erythrocyte conjugate prepared by the above method.

[0309] In some embodiments, the method for preparing antibody-erythrocyte conjugates of the present invention can also be extended to other drug protein molecules-erythrocyte conjugates, such as fusion protein-erythrocyte conjugates.

[0310] IV. Treatment

[0311] In one aspect, the present invention provides pharmaceutical compositions or formulations comprising antibody-erythrocyte conjugates. These compositions or formulations may also optionally comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0312] In one aspect, the present invention provides a blood preparation comprising an antibody-erythrocyte conjugate of the present invention, such as a human blood preparation. In some embodiments, the human blood preparation of the present invention comprises 10-1000 μg / mL of antibody-erythrocyte conjugate, such as 10-500 μg / mL of antibody-erythrocyte conjugate, for example, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL or more, or within any range of the numerical value. In some embodiments, the blood preparation of the present invention is a leukopenia blood preparation, i.e., a blood preparation in which leukocytes have been filtered out. In some embodiments, the blood preparation of the present invention is a human blood leukopenia blood preparation.

[0313] In some embodiments, the blood preparation of the present invention can be an allogeneic blood preparation, such as an allogeneic human blood preparation. In some embodiments, the red blood cells in the blood preparation are from healthy subjects. In some embodiments, the red blood cells in the blood preparation can be mature red blood cells differentiated from stem cells (e.g., obtained by culturing stem cells to differentiate into mature red blood cells).

[0314] In some embodiments, the blood preparation of the present invention can be an autologous blood preparation, such as an autologous human blood preparation. In some embodiments, the red blood cells in the blood preparation are from the subject to be treated.

[0315] In one aspect, the present invention also provides a combination product (eg, a pharmaceutical combination product) comprising an antibody-erythrocyte conjugate of the present invention and one or more other therapeutic agents. The combination product of the present invention can be used in the treatment methods of the present invention.

[0316] The present invention also provides a kit comprising the combination product, for example, the kit comprises in the same package:

[0317] - a first container containing the antibody-erythrocyte conjugate of the present invention or a pharmaceutical composition or formulation comprising the same;

[0318] - A second container comprising one or more additional therapeutic agents or a pharmaceutical composition or formulation comprising the same.

[0319] In some embodiments, the therapeutic agent is selected from any substance effective in cancer, including chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, small molecule drugs or immunomodulators (such as immunosuppressants or immunoagonists). Preferably, the therapeutic agent is selected from tumor vaccines, immune checkpoint inhibitor antibodies or immune agonist antibodies.

[0320] In one aspect, the present invention relates to a method for preventing or treating a disease, such as a tumor, in a subject, comprising administering to the subject an effective amount of an antibody-erythrocyte conjugate as described herein. In some embodiments, the tumor is cancer.

[0321] The cancer may be in the early, middle, or late stages or may be metastatic. In some embodiments, the cancer may be a solid tumor or a blood tumor. In some embodiments, the cancer is a gastrointestinal tumor, such as colon cancer or colorectal cancer; or it is lung cancer, pancreatic cancer, or breast cancer. In some embodiments, the tumor is resistant to or insensitive to immune checkpoint blockade therapy. In some embodiments, the immune checkpoint blockade therapy refers to treatment with an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an antibody that targets an immune checkpoint. In some embodiments, the immune checkpoint is selected from PD1, PD-L1, or PD-L2. In some embodiments, the tumor is sensitive to or insensitive to PD-1 and / or PD-L1 immunotherapy. In some embodiments, the tumor is resistant to or insensitive to PD-1 and / or PD-L1 immunotherapy. In some embodiments, the PD1 and / or PD-L1 immunotherapy refers to treatment with a PD-1 or PD-L1 inhibitor. In some embodiments, the tumor is a tumor or cancer that is resistant or insensitive to a known drug, such as a known PD-1 inhibitor or PD-L1 inhibitor, such as an anti-PD-1 antibody or an anti-PD-L1 antibody, such as a refractory tumor or cancer.

[0322] In some embodiments, the cancer is characterized as having elevated protein levels and / or nucleic acid levels of PD-1, PD-L1 and / or PD-L2 (e.g., elevated expression), for example, elevated protein levels and / or nucleic acid levels of PD-1, PD-L1 and / or PD-L2 in tumor cells (e.g., elevated expression), for example, compared to the protein levels and / or nucleic acid levels of PD-1, PD-L1 and / or PD-L2 in normal cells in corresponding tissues in a healthy individual, or compared to the protein levels and / or nucleic acid levels of PD-1, PD-L1 and / or PD-L2 in normal cells in adjacent healthy tissues of the same individual.

[0323] In some embodiments, the antibody-erythrocyte conjugates of the present invention can be used to stimulate the host's immune system, such as to enhance cellular immune responses. "Stimulating the immune system" can include any one or more of an overall increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, an increase in T cell activity or natural killer cell activity or lymphokine-activated killer (LAK) cell activity, an increase in T cell or natural killer cell survival, an increase in expression of cell-killing effector proteins, and the like.

[0324] The antibody-erythrocyte conjugates of the present invention (as well as compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same, such as blood preparations) can be administered by any suitable method, preferably by infusion, such as parenteral infusion. Parenteral infusion includes intravenous or intraarterial administration. Preferably, the antibody-erythrocyte conjugates of the present invention are administered by intravenous or intraarterial infusion.

[0325] For the prevention or treatment of disease, the appropriate dosage of the antibody-erythrocyte conjugates of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the administration is for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient as a single treatment or over a series of treatments.

[0326] In some embodiments, the antibody-erythrocyte conjugate of the present invention, or a pharmaceutical composition or preparation comprising the same, is administered in combination with one or more therapies such as treatment modalities or other therapeutic agents. In some embodiments, the treatment modality is radiotherapy or surgical treatment. In some embodiments, the therapeutic agent is selected from any substance effective in cancer, including chemotherapeutics, other antibodies, cytotoxic agents, vaccines, small molecule drugs or immunomodulators (such as immunosuppressants or immune agonists), preferably, the therapeutic agent is selected from tumor vaccines, immune checkpoint inhibitor antibodies or immune agonist antibodies. In some embodiments, the therapy is immunotherapy. In some embodiments, the therapy is cell therapy.

[0327] In another aspect, the present invention also provides use of the antibody-erythrocyte conjugate of the present invention or a pharmaceutical composition comprising the same in preparing a medicament or preparation for the aforementioned method (eg, for treatment).

[0328] In some embodiments, the present invention relates to an antibody-erythrocyte conjugate of the present invention or a pharmaceutical composition comprising the same for use in therapy, eg, for treating a disease described herein.

[0329] In some embodiments, the red blood cells in the antibody-red blood cell conjugates of the present invention are from the subject to be treated. In some embodiments, the red blood cells in the antibody-red blood cell conjugates of the present invention are autologous red blood cells. In some embodiments, the method of treating a disease in a subject of the present invention comprises:

[0330] (1) Collecting blood from the subject to be treated;

[0331] (2) preparing an antibody-erythrocyte conjugate, for example, by the method of the present invention;

[0332] (3) Infusing a therapeutically effective amount of the antibody-erythrocyte conjugate into the subject.

[0333] In some embodiments, the red blood cells in the antibody-erythrocyte conjugates of the present invention can be from other subjects, such as healthy subjects. In some embodiments, the red blood cells in the antibody-erythrocyte conjugates of the present invention are allogeneic red blood cells, such as allogeneic red blood cells from healthy subjects. In some embodiments, the antibody-erythrocyte conjugates of the present invention can be administered to the subject to be treated as a medicament or formulation.

[0334] In some embodiments, the antibody-erythrocyte conjugate drug or preparation and the treatment methods and uses thereof described in the present invention can also be extended to other drug protein molecule-erythrocyte conjugates, such as fusion protein-erythrocyte conjugates.

[0335] Any documents cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entirety.

[0336] Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner and are not intended to and should not limit the scope of the present invention in any way, and that various modifications may be made by those skilled in the art. Example:

[0337] Example 1 Preparation of PD-1-targeted red blood cells

[0338] 1. Preparation of Engineered Red Blood Cells (Taking the Reaction of 100 μL of Concentrated RBCs as an Example)

[0339] 1.1 Obtaining red blood cell intermediates

[0340] Open the cap of the blood collection tube and use a disposable syringe to transfer the human blood sample from the healthy subject into a blood bag. Once completed, seal the tube twice with a sterile tube sealer. Leukocytes were removed from the whole blood using a leukocyte filter. Using a 20 mL disposable syringe, transfer the leukocyte-removed blood sample from the blood storage bag into a 50 mL centrifuge tube and centrifuge to collect red blood cells. Centrifugation parameters were: 500 g centrifugation force, 5 minutes of centrifugation, 9 ramps, 8 ramps, and 25°C.

[0341] Centrifuge to obtain the concentrated red blood cell intermediate. The centrifugation parameters are set at 500 g for 3 minutes, a ramp rate of 9, a ramp rate of 7, and a temperature of 25°C. Open the centrifuge tube and discard the supernatant using a disposable sterile pipette or a 1000 μL pipette with a matching tip. Aspirate the concentrated red blood cells using an electric pipette and add them to the reaction vessel.

[0342] 1.2 Antibody protein processing

[0343] 1.2.1 Preparation of Sulfo-SMCC

[0344] Take out Sulfo-SMCC powder (purchased from Thermo Fisher, product number 22322) from the -20 degree refrigerator, accurately weigh 2 mg and place it in an EP tube, add 2 ml PBS, and shake to completely dissolve it. The concentration after dissolution is 1 mg / ml.

[0345] 1.2.2 Antibody protein treatment

[0346] Take 1mg / ml Sulfo-SMCC solution, draw 21.8μL and add it to 138.2μL PBS buffer, and mix it evenly by pipetting gently. Draw 40μL anti-PD-1 antibody solution ( Add MSD to the EP tube and mix thoroughly to achieve a final concentration of 0.25 mM Sulfo-SMCC. Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 hour to obtain the treated anti-PD-1 antibody protein solution.

[0347] After the reaction is completed, add the treated PD-1 antibody protein solution to the inner tube of the ultrafiltration tube, add PBS buffer to the white line at the top of the ultrafiltration tube and centrifuge. After the centrifugation is completed, remove the upper ultrafiltration tube, pour out the waste liquid in the lower tube, reinstall the upper ultrafiltration tube, add PBS buffer to the white line at the top of the ultrafiltration tube, mix well and centrifuge. Centrifuge until the upper volume is less than or equal to 100 μL (theoretical concentration of anti-PD-1 antibody protein solution ≥10 mg / mL or ≥35 mg / mL). After the centrifugation is completed, aspirate the upper anti-PD-1 antibody protein solution and add PBS to 100 μL (so that the theoretical concentration of anti-PD-1 antibody protein solution is 10 mg / mL). Centrifugation parameters: centrifugal force 3800g, acceleration 8, deceleration 8, temperature 4°C.

[0348] 1.3 Surface modification of red blood cells

[0349] 1.3.1 Preparation of 0.1M TCEP

[0350] Remove an ampoule of TCEP stock solution (tris-(2-carboxyethyl)phosphine hydrochloride, purchased from Sigma-Aldrich, Cat. No. 646547) labeled at 0.5M from a -20°C refrigerator and place on ice until the solution in the ampoule is completely melted. After surface disinfection, place in a biosafety cabinet and let it stand for 1 minute. Carefully open the ampoule and add 100μL of TCEP stock solution to a 2mL EP tube. Then add 400μL of PBS and pipette to mix thoroughly to prepare a TCEP stock solution dilution. Use a 1mL syringe to draw up the TCEP stock solution dilution and filter through a 0.22μm filter to sterilize, obtaining a 0.1M TCEP sterile solution. Aliquot 20μL of the sterile TCEP solution into sterile 1.5mL EP tubes. Seal the tubes, attach sample labels, and store in a -20°C refrigerator until ready for use.

[0351] 1.3.2 Red blood cell surface modification

[0352] Pipette 10 μL of 0.1 M TCEP into the EP tube containing 90 μL of PBS buffer and mix thoroughly. Pipette 100 μL of the concentrated red blood cells obtained in 1.1 above and add them to the solution. Mix thoroughly to a final TCEP concentration of 5.0 mM. Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 hour.

[0353] After the reaction is complete, centrifuge and remove the supernatant. Add PBS buffer (5 times the volume of the concentrated red blood cells), mix thoroughly, and centrifuge. Discard the supernatant. Repeat this step twice. Centrifugation parameters: 800g, 3 minutes, 9% ramp rate, 7% ramp rate, 25°C. Collect the modified concentrated red blood cells.

[0354] 1.4 Red blood cell engineering

[0355] Add 100 μL of modified concentrated RBC to 100 μL of ultrafiltered anti-PD-1 antibody protein solution, gently pipette to mix evenly, and place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 h.

[0356] After the reaction is complete, centrifuge and remove the supernatant. Add 9 times the volume of physiological saline to the concentrated RBC volume, mix thoroughly, and centrifuge. Remove the supernatant. Repeat this step twice. Centrifuge parameters: 800 g, 3 minutes, 9% ramp rate, 7% ramp rate, and 25°C. Collect the engineered RBCs.

[0357] 2. Storage of Engineered Red Blood Cells

[0358] Pipette 100 μL of saline and 50 μL of red blood cell preservation solution (normal saline equal to the volume of concentrated red blood cells and 0.5 times the volume of MAP red blood cell preservation solution (Shandong Weigao Group)), add to the engineered red blood cells, and gently pipette to mix thoroughly to obtain the engineered red blood cell preparation solution. Add the engineered red blood cell preparation solution to the packaging container. After packaging, store at 4°C until ready to use.

[0359] Hereinafter, the engineered red blood cells coupled with Keytruda prepared in this example will be referred to as RBCs-Keytruda or RBC-Keytruda. Example 2 In vitro activity of engineered red blood cells

[0360] PD-1 / PD-L1 Blockade Bioassay

[0361] 1. Experimental principle:

[0362] When the two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR signaling and NFAT-mediated luciferase activity. Adding antibodies that block either PD-1 or PD-L1 relieves the inhibitory signal, leading to activation of the TCR signaling pathway and enhanced NFAT-mediated luciferase activity.

[0363] 2. Experimental Materials

[0364] PD-1 effector cells: Jurkat T cells (Promega, J115A), stably expressing human PD-1 and NFAT-inducible luciferase.

[0365] PD-L1 aAPC / CHO-K1 cells (Promega, J109A): CHO-K1 cells stably express human PD-L1 and a cell surface protein that can activate the cognate TCR in an antigen-independent manner.

[0366] 3. Experimental Equipment

[0367] IV. Experimental consumables

[0368] 5. Experimental Reagents

[0369] 6. Experimental Procedure

[0370] 1. Cell Line Preparation

[0371] Cell digestion: Take T75 culture flask as an example. Add 1mL of trypsin and place the cell culture flask in a CO2 incubator for about 2-3 minutes to allow the cells to fall off the surface of the cell flask. Add growth medium to the culture flask to terminate digestion and use a pipette to blow the cells off. Transfer the cell suspension in the culture flask to the same disposable sterile centrifuge tube. Centrifuge at 1000rpm for 5 minutes. Discard the supernatant and add 10mL of growth medium to resuspend the cells. Use a cell counter to determine the cell density and cell viability. Adjust the cell density to 4×10 per milliliter. 5 Add 100 μL of cell suspension to a 96-well white flat-bottom assay plate, with a final concentration of 40,000 cells per well.

[0372] Place the assay plate in a CO2 incubator overnight (16-20 hours).

[0373] 2. Detection Method

[0374] Serial dilution of PD-1 antibody and red blood cell preparation samples: Prepare fresh assay buffer (98% PRMI 1640 + 2% FBS) on the day of the assay. Determine the required volume of assay buffer based on the number of samples. Dilute PD-1 antibody (98% PRMI 1640 + 2% FBS) with assay buffer. Perform a 10-point 3-fold serial dilution using MSD (MSD), with the highest dilution point being 5 μg / mL. The sample volume at each step should be no less than 10 μL. Vortex mix thoroughly after each dilution step. If the sample concentration is higher than 1 mg / mL, the dilution factor at each step should not exceed 100-fold.

[0375] Prepare Jurkat-PD-1 Effector cells: Subculture Jurkat T cells before testing and culture to a density of 1×106 cells / mL. Note that the cell viability must be greater than 80%. Cell collection: Transfer the supernatant from the cell culture flask to a 50mL disposable sterile centrifuge tube. Use the assay buffer at a concentration of 1.25×10 6 Resuspend the cells at a density of 1 / mL.

[0376] Add antibodies or red blood cell preparations and Jurkat-PD-1 Effector cells to the assay plate: Add 40 μl of serially diluted red blood cell preparations or PD-1 antibodies to the corresponding wells. Add assay buffer to the blank control wells. Add 40 μl of Jurkat-PD1 Effector cells to the wells containing PD-L1 aAPC / CHO-K1 cells and antibodies or red blood cell preparations and the blank control wells. Incubate the assay plate in a 37°C / 5% CO2 incubator for 4-6 hours. Bright- Luciferase Assay Adding Bright- Reagent. Add 40 μL of right- Add the detection reagent and incubate at room temperature for 8-15 minutes. Detect the signal using a microplate reader or a luminescence detection instrument.

[0377] 7. Data Analysis

[0378] Sample concentration calculation method: Data processing software is used to calculate and analyze data. Calculate the %CV value of the test sample and reference sample in duplicate. With the concentration of the standard as the X-axis and the induction factor as the Y-axis, use a four-parameter regression model to draw a standard curve and calculate the R2 of the fitted curve. y = D + (AD) / (1 + (X / C)^B)

[0379] The relative activity of the sample is calculated as follows: Relative binding activity = EC50 value of the reference product / EC50 value of the test product * 100%

[0380] The calculation formula of relative standard deviation is as follows: RSD = (SD / AVERAGE) × 100%

[0381] Parallelism slope ratio = test sample B value / reference sample B value

[0382] Upper asymptote ratio D value ratio = test sample D value / reference sample D value

[0383] 8. Experimental Results

[0384] The experimental results are shown in Figures 1B and 1C. The PD-1 / PD-L1 blocking bioassay results show that at a low dose of Keytruda, RBC-Keytruda can produce an in vitro agonist effect more quickly (Figure 1B). At the same dose, the RBC-Keytruda formulation can significantly enhance the efficacy of the PD1 antibody (Figure 1C).

[0385] Example 3 SK-RBC treatment of MC38 model

[0386] 1. Research objectives:

[0387] The purpose of this study was to evaluate the tumor killing function of mouse engineered red blood cells coupled with Keytruda in the MC38 cell tumor model in PD-1 humanized mice.

[0388] 2. Research Methods

[0389] Mouse colon cancer MC38 cells (Jicui Yaokang) were cultured at 5×10 5 Cell numbers were injected subcutaneously into PD1 humanized mice.

[0390] In this mouse model, all experimental animals were divided into 4 groups:

[0391] (1) Control group, blank PBS;

[0392] (2) Keytruda low-dose treatment group;

[0393] (3) Keytruda high-dose treatment group;

[0394] (4) RBCs-Keytruda treatment group.

[0395] When the mouse tumor size is about 50mm 3 The patients were randomly divided into groups and drug administration was started. The tumor volume was measured every 3-4 days. 3 The mice were killed at 4 pm and the tumor volumes of mice in each group were compared.

[0396] Experimental Materials:

[0397] 2.1 Preparation of Mouse Engineered Red Blood Cells Coupled with Keytruda

[0398] Mouse engineered red blood cells coupled to Keytruda were prepared similarly to Example 1, wherein

[0399] Sulfo-SMCC treated with 5mg Keytruda (mol ratio 9.3:1)

[0400] Take out a Sulfo-SMCC from the -20 degree refrigerator, add 2 ml of PBS, and shake to dissolve it completely. The concentration after dissolution is 1 mg / ml.

[0401] Take a 2.0ml EP tube and add sample according to the following table:

[0402] After reacting at 10 rpm for 1 hour, ultrafiltration was performed twice using a 50 KDa ultrafiltration tube at 3800 g for 15 minutes, and the solution was quantified to 250 μl.

[0403] TCEP treatment of 1.5 ml concentrated mRBC

[0404] Centrifuge 3 ml of whole blood from adult PD1 humanized C57BL / 6 mice at 1000 g / 5 min for three minutes and discard the supernatant. Transfer the concentrated RBCs to a 15 ml centrifuge tube, fill with PBS, and centrifuge twice at the same speed and time to wash. Discard the supernatant. Add samples according to the table below.

[0405] After reacting at 10 rpm for 1 hour, mRBCs were washed twice with PBS using the above method and the supernatant was discarded.

[0406] The ultrafiltered Keytruda was mixed with 500ul RBC (without PBS), mixed at 10 rpm for 1 hour, and then washed twice with PBS.

[0407] The sample was labeled with FC flow cytometry antibody at a dilution of 1:200 for 2 minutes and then subjected to flow cytometry detection. The sample was aliquoted after the coupling efficiency reached 100%.

[0408] The prepared blood samples were divided into smaller portions according to a 1:5 concentration gradient.

[0409] Take 416 μl of RBCs-Keytruda and add PBS to make up to 1.25 ml. Label the tube "RBCs-Keytruda."

[0410] Prepare 1.25 ml of 0.005 mg / ml Keytruda in PBS. Label the container "Keytruda-low"

[0411] Prepare 1.25 ml of 0.05 mg / ml Keytruda in PBS. Label the tube as "Keytruda-high"

[0412] Take 1.25 ml of PBS and label it "Control PBS".

[0413] 2.2 Experimental methods

[0414] Experimental animals:

[0415] The experimental animals used were PD1 humanized C57BL / 6 female mice purchased from Jicui Yaokang. The initial experimental age was 6 to 8 weeks.

[0416] Dosage method and group design:

[0417] Route and method of administration: Tail vein injection

[0418] Reasons for choosing the route of administration: The transfusion method of engineered red blood cell products should refer to the transfusion method of concentrated red blood cell products in the "Guidelines for Whole Blood and Blood Component Quality Monitoring".

[0419] Dosage frequency and duration: Once every three days, for a total of 6 injections.

[0420] Administration time: 16:00-17:00

[0421] Dosage volume: 200 μL

[0422] Administration rate: 25 μL / s, controlled by syringe administration.

[0423] The tumor cell line selected was MC38 cells, which can form tumors subepithelially in C57BL / 6 mice.

[0424] 3. Experimental Results

[0425] The results are shown in Figure 2. Statistical analysis of tumor volume data during the efficacy phase revealed that, compared with the G1 (PBS) control group, the Keytruda group (G2, 0.05 mg / kg) showed no significant tumor growth inhibition. The Keytruda group (G3, 0.5 mg / kg) showed significant tumor growth inhibition starting on D7 (day 7) after administration, reaching a peak tumor inhibition rate of 78.02% on D24 (day 24) with statistical significance (**P<0.01). Subsequently, the TGI gradually declined, reaching a tumor inhibition rate of 75.53% on D30 (day 30).

[0426] The RBCs-Keytruda group (G4, 0.5 mg / kg) showed significant tumor growth inhibition starting on D7 after administration, reaching a peak tumor inhibition rate of TGItv = 91.09% on D27 (day 27) of observation, with a statistically significant difference (***P<0.001). Subsequently, TGI gradually declined, with a tumor inhibition rate of TGItv = 90.44% on D30 (day 30). There was no significant statistical difference in body weight changes among the mice treated with mouse engineered red blood cells coupled to Keytruda. In summary, the high-dose administration group of mouse engineered red blood cells coupled to Keytruda had a significant tumor inhibitory effect on mice bearing MC38 mouse colon cancer cell lines.

[0427] TGITV (relative tumor inhibition rate) calculation formula:

[0428] V nt : Tumor volume of mouse numbered n on day t;

[0429] V n0 : Tumor volume of mice numbered n on day 0;

[0430] RTV n : Relative tumor volume of mouse numbered n on day t;

[0431] mean RTV treat : Mean RTV value of the drug-treated group;

[0432] mean RTV vehicle : Mean RTV value of the control group (Group 1);

[0433] Tumor volume was measured with a caliper.

[0434] Example 4 SK-RBC treatment of KP model

[0435] 1. Research objectives:

[0436] The purpose of this study was to evaluate the tumor killing function of mouse engineered red blood cells coupled to Keytruda (RBC-Keytruda, prepared as described in Example 3) in a KP cell tumor-bearing model in PD-1 humanized mice.

[0437] 2. Research Methods

[0438] Mouse lung cancer KP (KRAS G12D, P53- / -) cells (Shanghai Cell Bank) were cultured at 1×10 6 The cells were injected subcutaneously into mice. In this mouse model, all experimental animals were divided into 3 groups: (1) control group, blank RBC; (2) RBC-Keytruda high-dose treatment group; (3) Keytruda alone treatment group. When the mouse tumor size was about 50mm 3 The patients were randomly divided into groups and drug administration was started. The tumor volume was measured every 3-4 days. 3 The mice were killed at 4 pm and the tumor volumes of the two groups were compared.

[0439] 3. Experimental Animals

[0440] The experimental animals used were PD1 humanized female C57BL / 6 mice purchased from Jicui Yaokang. The initial experimental age was 6 to 8 weeks.

[0441] 4. Animal Dosing Method

[0442] Route and method of administration: Tail vein injection

[0443] Reasons for choosing the route of administration: The transfusion method of engineered red blood cell products should refer to the transfusion method of concentrated red blood cell products in the "Guidelines for Whole Blood and Component Blood Quality Monitoring".

[0444] Dosage frequency and duration: Once every three days, for a total of 6 injections.

[0445] Administration time: 4:00 PM to 5:00 PM

[0446] Dosage volume: 200μL

[0447] Administration rate: 25 μL / s, controlled by syringe administration.

[0448] 5. Grouping of Experimental Animals

[0449] The tumor cell line selected was KP cells, which can form tumors subepithelially in C57 / BL6 mice.

[0450] 6. Experimental Results

[0451] As shown in Figure 3, the survival status of mice was monitored at different times. Due to the poor condition of the mice in the Keytruda alone treatment group, all mice died on day 31 of the experiment. However, all mice in the control group and the RBC-Keytruda treatment group were still alive after 39 days of the experiment. In vivo pharmacokinetics showed that after administration of Keytruda, the peripheral blood content was high, resulting in certain tissue toxicity, such as intestinal toxicity. However, after administration of the RBC-Keytruda of the present invention, the peripheral blood content was low and mainly distributed in the blood vessels and liver and spleen systems, thus, the tissue toxicity was relatively small.

[0452] As shown in Figure 4, the tumor volume of mice treated with mouse engineered red blood cells coupled to Keytruda RBC-Keytruda was measured with a caliper 39 days after KP tumor cell injection. The average tumor size of the control group mice was 1103.95 mm 3 The average tumor size of mice in the RBC-Keytruda group was 488.32 mm 3 The tumor size in the drug-treated group was significantly smaller than that in the control group (**p<0.01), and the tumor growth inhibition rate (TGItw) was 55.76%.

[0453] Tumor growth inhibition rate (TGItw) calculation formula:

[0454] TGItw=(1-meanTW treat / meanTW vehicle)x100%

[0455] meanTWtreat: the average tumor weight of mice in the treatment group at the end point of treatment

[0456] meanTW vehicle: the mean tumor weight of mice in the control group at the end of treatment

[0457] As shown in Figure 5, in mice treated with engineered red blood cells coupled to Keytruda, the total number of tumor-infiltrating CD8+ cells in the RBC-Keytruda group was 2.90 times higher than in the control group 39 days after KP tumor cell injection, indicating that RBC-Keytruda can effectively activate the immune system.

[0458] Therefore, engineered red blood cells coupled with Keytruda have a significant tumor inhibitory effect on KP tumor cell line PD-1 humanized mice.

[0459] Example 5 SK-RBC treatment of PB3 model

[0460] 1. Research objectives:

[0461] The purpose of this study was to evaluate the tumor killing function of mouse engineered red blood cells coupled with Keytruda in a PB3 cell tumor model in PD-1 humanized mice.

[0462] 2. Research Methods

[0463] Mouse breast cancer PB3 cells (Epithelial-to-Mesenchymal Transition Contributes to Immunosuppression in Breast Carcinomas, Anushka Dongre, Cancer Res. 2017 Aug 1; 77(15): 3982-3989. doi: 10.1158 / 0008-5472.CAN-16-3292. Epub 2017 Apr 20.) were cultured at a medium temperature of 5×10 4 The cells were injected subcutaneously into mice. In this mouse model, all experimental animals were divided into 3 groups:

[0464] (1) Control group, blank PBS;

[0465] (2) Keytruda alone treatment group;

[0466] (3) RBCs-Keytruda treatment group.

[0467] When the mouse tumor size is about 100mm 3 At 3 pm, the patients were randomly divided into groups and drug administration began.

[0468] Tumor volumes were measured with a caliper every three days, and all mice were sacrificed after four doses of administration. The tumor volumes of the two groups of mice were compared.

[0469] 3. Materials and Methods

[0470] 3.1 Preparation of Mouse Engineered Red Blood Cells Coupled with Keytruda

[0471] Mouse engineered red blood cells coupled to Keytruda were prepared as described in Example 3, except that the prepared blood samples were divided into aliquots according to a 1:5 concentration gradient.

[0472] ① Take 500 μl of RBCs-Keytruda and dilute to 1.25 ml with PBS. Label the tube "RBCs-Keytruda."

[0473] ② Take 1.25 ml of PBS and label it "Control PBS".

[0474] ③Take 1213ul PBS, add 47ul Keytruda, and mark the name "Keytruda".

[0475] 3.2 Experimental methods

[0476] experimental animals

[0477] The experimental animals used were PD1 humanized female C57BL / 6 mice purchased from Jicui Yaokang. The initial experimental age was 6 to 8 weeks.

[0478] Animal grouping and drug administration

[0479] 3.2.1 Animal Dosing Method

[0480] Route and method of administration: Tail vein injection

[0481] Reasons for choosing the route of administration: The transfusion method of engineered red blood cell products should refer to the transfusion method of concentrated red blood cell products in the "Guidelines for Whole Blood and Component Blood Quality Monitoring".

[0482] Dosage frequency and duration: Once every three days, for a total of 4 injections.

[0483] Administration time: 4:00 PM to 5:00 PM

[0484] Dosage volume: 200μL

[0485] Administration rate: 25 μL / s, controlled by syringe administration.

[0486] 3.2.2 Experimental Animal Grouping

[0487] The tumor cell line selected was PB3 cells, which can form tumors subepithelially in C57BL / 6 mice.

[0488] 4. Experimental Results

[0489] As shown in Figure 6 (tumor volume change curve) and Figure 7 (key tumor weight), the tumor size of mice treated with mouse engineered red blood cells coupled with Keytruda was weighed at the end point of PB3 model treatment. The RBCs-Keytruda treatment group showed a significant anti-tumor effect (TGItw = 36.46%), and there was a significant statistical difference (*P < 0.01), while the Keytruda alone treatment group had no significant anti-tumor effect (TGItw = -0.55%).

[0490] 5. Experimental Conclusion

[0491] The mouse engineered red blood cells coupled with Keytruda had a significant tumor inhibitory effect on mice bearing PB3 mouse breast cancer cell lines.

[0492] Example 6 RBC-Keytruda Pharmacokinetics

[0493] I. Engineered red blood cells can reach the spleen

[0494] Immunohistochemical staining experiments confirmed that RBC-Keytruda could reach the spleen. The experimental process is as follows:

[0495] 1. Use 6-8 weeks old PD1 humanized mice (purchased from Jicui Yaokang), 1x10 e6 Mouse lung cancer cell line KP (Shanghai Cell Bank) was injected subcutaneously to form tumors;

[0496] 2. When the tumor grows to 100 mm 3 At the same time, Far-red dye (purchased from Thermo, CellTrace) was injected into the tail vein of two mice. TM ) labeled 200ul mouse control red blood cells (RBCs-Control) and 200ul engineered red blood cells prepared in Example 1 (coupled with Keytruda (RBCs-Keytruda));

[0497] 3. 24 hours after administration, the mice were killed, and spleen tissues were obtained for frozen sections;

[0498] 4. Immunofluorescence staining of spleen sections from two mice using Fc antibody (Thermo, cat. no. 12-4998-82);

[0499] 5. Photographed under confocal microscope, Far-red is the far infrared channel (red), and Keytruda is the Fc staining channel (green). The experimental results are shown in FIG1A , indicating that the RBCs-Keytruda of the present invention can reach the spleen.

[0500] II. RBC-Keytruda Pharmacokinetics

[0501] 1. Research objectives:

[0502] The purpose of this study was to evaluate the in vivo pharmacokinetic profile of Keytruda conjugated to mouse engineered red blood cells in mice.

[0503] 2. Research Methods

[0504] The Keytruda-coupled mouse engineered red blood cells were stained with the cell dye Cell Trace Far Red and the cells were centrifuged at 1×10 9The cells were injected into C57BL / 6 mice via the tail vein. In this experiment, the treated engineered red blood cells were injected into three mice, with the injection time recorded as day 0. Peripheral blood was collected from the mice on days 1, 3, 7, 14, and 21. Flow cytometry was used to analyze the proportion of Cell Trace Far Red-positive cells in the mouse peripheral blood and the proportion of Keytruda-positive cells in Cell Trace Far Red-positive cells. The retention of engineered red blood cells in the body and the retention of Keytruda on the engineered red blood cells at different time points were detected.

[0505] 3. Materials and Methods

[0506] 3.1 Preparation of Mouse Engineered Red Blood Cells Coupled with Keytruda

[0507] Prepare Keytruda-conjugated mouse engineered red blood cells (RBCs-Keytruda) as described in Example 3. Take 416 μl of RBCs-Keytruda from the prepared blood sample and add PBS to a volume of 1.25 ml. Label the sample "RBCs-Keytruda."

[0508] The RBCs-Keytruda fixed to volume with PBS were stained with Cell Trace Far Red (Thermo) as follows:

[0509] RBCs-Keytruda cells were centrifuged at 1000×g for three minutes, the supernatant was removed, and the cells were resuspended in PBS solution;

[0510] Add Cell Trace Far Red dye at a dilution of 1:1000 and gently resuspend the cells;

[0511] The cells were incubated at 37°C in the dark for 20 minutes;

[0512] The reaction system was terminated by adding PBS containing serum and incubated at 37°C in the dark for 5 minutes;

[0513] The cells were centrifuged at 1000×g for three minutes, the supernatant was removed, and the cells were resuspended in PBS solution for later use.

[0514] 3.2 Experimental methods

[0515] experimental animals

[0516] The experimental animals used were C57BL / 6 mice purchased from the Experimental Animal Center of Westlake University. The initial experimental age was 6 to 8 weeks.

[0517] 3.2.1 Animal Dosing Method

[0518] Route and method of administration: Tail vein injection

[0519] Reasons for choosing the route of administration: The transfusion method of engineered red blood cell products should refer to the transfusion method of concentrated red blood cell products in the "Guidelines for Whole Blood and Component Blood Quality Monitoring".

[0520] Administration time: 4:00 PM to 5:00 PM

[0521] Dosage volume: 200μL

[0522] Administration rate: 25 μL / s, controlled by syringe administration.

[0523] 4. Experimental results:

[0524] During the pharmacokinetic process, flow cytometry was used to analyze the proportion of Cell Trace Far Red-positive cells in mouse peripheral blood (Figure 8) and the proportion of Keytruda-positive cells in Cell Trace Far Red-positive cells (Figure 9). The proportion of mouse engineered red blood cells coupled to Keytruda decreased rapidly from D0 to D3 in the mouse body, and then slowly decreased from D3 to D21 until the red blood cells were completely cleared. Analysis of Keytruda retention on engineered red blood cells showed that all Cell Trace Far Red-positive cells were Keytruda-positive from D0 to D21.

[0525] 5. Experimental conclusion:

[0526] Engineered red blood cells coupled to Keytruda can remain in the mice's body for up to 21 days and are eventually cleared along with the mice's engineered red blood cells.

[0527] Example 7 Comparison of drug loading and relative activity of anti-PD-1-RBC preparations prepared with different brands of anti-PD-1 antibodies

[0528] 1. Purpose of the experiment

[0529] Compare the drug loading and relative activity of anti-PD-1-RBC preparations prepared using the process of this project using different brands of PD-1 antibodies.

[0530] 2. Experimental Methods

[0531] 2.1 Preparation of anti-PD-1-RBC antibodies of different brands

[0532] Antibody brand names (abbreviations): Hengrui (HR), BeiGene (BJ), Innovent (XD), Merck (K)

[0533] Referring to Example 1, different brands of anti-PD-1 antibodies SMCC-HR, SMCC-BJ, SMCC-XD and SMCC-K linked to SMCC, as well as anti-PD-1-RBC preparations (K-hRBC-211227, BJ-hRBC-211227, HR-hRBC-211227 and XD-hRBC-211227) of human blood RBCs coupled to different brands of anti-PD-1 antibodies were prepared.

[0534] With reference to Example 3, anti-PD-1-RBC preparations K-mRBC-211227, BJ-mRBC-211227, HR-mRBC-211227 and XD-mRBC-211227 of mouse blood RBCs coupled with different brands of anti-PD-1 antibodies were prepared.

[0535] 2.2 In vitro relative activity detection methods for antibodies and preparations

[0536] Refer to the test method of Example 2.

[0537] 2.3 Determination of drug loading of anti-PD-1-RBC preparations

[0538] 2.3.1 Experimental process

[0539] This experiment uses the ELISA method to detect the content of anti-PD-1 antibodies in the anti-PD-1-RBC preparation prepared in 1. The specific operation process is as follows:

[0540] 2.3.1.1 Test solution preparation

[0541] Preparation of antigen solution: Add 1 mL of sterile water to PD1 antigen dry powder (ACRO, PD1-H522) until completely dissolved to 25 mg / mL. Aliquot and store at -80°C to -60°C. Valid for 3 months.

[0542] Blocking solution: 2% BSA (Solybol, A8020)-PBS. Weigh 2.0 g of BSA and add it to 100 mL of PBS solution until completely dissolved. Store at 2°C-8°C. Valid for 7 days.

[0543] Washing solution: PBST, 1×PBS plus 0.05% Tween 20 (Sanggong, A600560-0500), mix well, store at room temperature, valid for 30 days.

[0544] Sample diluent: 0.1% BSA-PBST solution. Weigh 0.1g of BSA and add it to 100mL of 0.1% PBST until completely dissolved. Alternatively, add 1mL of 2% BSA-PBS to 19mL of 0.1% PBST and mix thoroughly. Store at 2°C-8°C. Expiration date: 7 days.

[0545] 2.3.1.2 ELISA test process

[0546] Coating: Dilute the above-prepared human PD-1 antigen solution (PD1 Leu 25-Gln 167 (Accession# NP_005009.2)) to 500 ng / mL with PBS, coat the plate, and incubate overnight at 2°C-8°C.

[0547] Wash the plate: Wash the plate 3 times with detergent.

[0548] Blocking: Add blocking solution to a 96-well plate and block at 37°C for 1-2 hours.

[0549] Sample addition: Take 16,000-fold and 32,000-fold diluted anti-PD-1-RBC preparations, as well as linearly diluted (0-2ug / mL) anti-PD-1 antibodies of various brands, and add 50μL each to a 96-well plate, place two parallel wells, and incubate at 37°C for 1h-2h.

[0550] Wash the plate: Wash the plate 4 times with detergent.

[0551] Add secondary antibody: dilute the secondary antibody (Abcam, ab97225) with PBS and add 100 μL per well of a 96-well plate.

[0552] Color development: Add 100 μL / well of a methanol solution of tetramethylbenzidine (TMB, SURMODICS, TMD-1000-01) and incubate at 37°C in the dark for 15-20 minutes.

[0553] Stop: Add 50 μL / well of stop solution (Solabo, CL058).

[0554] Detection reading: Read the optical density absorbance value at OD450nm-570nm using a microplate reader.

[0555] 2.3.2 Result calculation

[0556] The data were calculated and analyzed using data processing software. The mean and %CV of the OD values ​​of the duplicate wells of the test samples and standards were calculated. The concentration of the standard was used as the X-axis and the mean value of OD450-570nm was used as the Y-axis. A four-parameter regression model was used to draw the standard curve and calculate the R of the fitted curve. 2 (Formula see below).

[0557] y=D+(AD) / (1+(X / C)^B)

[0558] By fitting the curve, the average of the OD values ​​of duplicate wells is used to calculate the PD1 antibody concentration (μg / mL) in the standard and test samples.

[0559] 3. Experimental Results

[0560] 3.1 Determination of the relative activity of SMCC after reaction with different brands of antibodies.

[0561] The relative activity of the reactants after Sulfo-SMCC reacted with different brands of PD-1 antibodies was tested in vitro. The results showed that under the existing process conditions, the reaction between sulfo-SMCC and antibodies itself had little effect on the antibody activity.

[0562] 3.2 Determination of drug loading and relative activity of human blood preparations prepared from different brands of antibodies

[0563] Human blood preparations were prepared using different brands of PD-1 antibodies, and engineered red blood cells carrying PD-1 antibodies were all prepared, with a drug loading of 52.24 to 109.15 μg / mL.

[0564] 3.3 Determination of drug loading in mouse blood preparations prepared with different brands of antibodies

[0565] C57BL / 6 mouse blood preparations were prepared using different brands of PD-1 antibodies, and engineered red blood cells carrying PD-1 antibodies were all prepared, with drug loading amounts ranging from 50.84 to 274.33 μg / mL.

[0566] Example 8. Comparison of drug loading and relative activity of anti-PD-1-RBC preparations prepared with different brands of anti-PD-1 antibodies

[0567] 1. Purpose of the experiment

[0568] Compare the coupling efficiency, in vitro activity and drug loading of different brands of PD-1 antibodies when using the process method of this project to prepare anti-PD-1-RBC preparations.

[0569] 2. Experimental Methods

[0570] 2.1 Preparation and coupling efficiency of different brands of anti-PD-1-RBC antibodies

[0571] Referring to Example 3, different brands of anti-PD-1 antibodies linked to SMCC and anti-PD-1-RBC preparations of mouse blood RBCs coupled to different brands of anti-PD-1 antibodies were prepared. The specific brands and identifications of the anti-PD-1-RBC preparations are shown in the table below:

[0572] Antibody brand and source

[0573] The coupling efficiency of the detection antibody was similar to that of Example 3-2.1, wherein the sample was labeled with Goat anti-Human IgG Fc (PE anti-Human IgG Fc Secondary Antibody) (1:200) for 2 minutes and then subjected to flow cytometry. The flow channel was PE, and the coupling efficiency of each brand of PD1 antibody and red blood cells was detected, showing that the coupling efficiency was approximately 100% (Figure 11).

[0574] The samples were labeled with FC flow cytometry antibody at a dilution of 1:200 for 2 minutes and then flow cytometry was performed.

[0575] 2.2 In vitro activity detection methods for preparations

[0576] As described in Example 2 above, PD1 expressed on Jurkat-PD1 Effector Cells can bind to the ligand PD-L1 on PD-L1aAPC / CHO-K1Cells to inhibit signal generation. After the PD1 protein on various brands of red blood cell preparations binds to PD-L1, it can block the combination of PD-L1 and PD-1, thereby activating downstream pathways to generate signals.

[0577] The in vitro relative activity was tested according to the test method of Example 2, wherein the initial maximum concentration of the red blood cell preparation of each brand of antibody used was 250 ng / ml, and the red blood cell preparation was diluted 2.5 times.

[0578] The results are shown in FIG12 , which shows that the red blood cell-coupled PD1 antibody preparations (anti-PD-1-RBC preparations) all have significant in vitro activity.

[0579] 2.3 Pharmacokinetics of Anti-PD-1-RBC Preparations

[0580] Similar to the "II. RBC-Keytruda Pharmacokinetics" section in Example 6, the in vivo pharmacokinetics of mouse engineered red blood cells coupled to different brands of antibodies were evaluated in mice. The preparation, materials, and injection methods of each brand of red blood cell preparations are described in Example 6-II-3.

[0581] Flow cytometry was used to detect antibody Fc in mouse peripheral blood using a Goat anti-Human IgG Fc flow cytometry antibody. The theoretical value was 10%, and the detection points were 30 minutes after injection (marked 0) and D3 (day 3, marked 3). The results are shown in Figure 13, showing that the red blood cell-coupled PD1 antibody preparation can be stably present in mice.

[0582] 2.4 Determination of drug loading of anti-PD-1-RBC preparations

[0583] The content of anti-PD-1 antibodies in the prepared anti-PD-1-RBC preparation (drug loading of the preparation) was detected by ELISA method as in Example 7-2.3. The results are shown in the table below:

[0584] Human blood preparations were prepared using different brands of PD-1 antibodies, and engineered red blood cells carrying PD-1 antibodies were prepared with drug loading ranging from 27.3 to 162.7 μg / 10 10 cells.

[0585] Example 9: SMCC-Keytruda peptide mapping and coupling site analysis

[0586] Preparation of SMCC-Keytruda

[0587] Take 1mg / ml sulfo-SMCC solution, draw 21.8μL and add it to 138.2μL PBS buffer, and mix it evenly by blowing gently; draw 40μL anti-PD-1 antibody solution ( Add MSD to the EP tube and mix thoroughly to achieve a final concentration of 0.25 mM sulfo-SMCC. Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 hour to obtain the treated anti-PD-1 antibody protein solution.

[0588] After the reaction is complete, the upper layer of the ultrafiltration tube is aspirated, and the treated anti-PD-1 antibody protein solution is added. PBS buffer is added to the top white line of the ultrafiltration tube and centrifuged. After centrifugation, the upper ultrafiltration tube is removed, the waste liquid in the lower tube is poured out, the upper ultrafiltration tube is replaced, and PBS buffer is added to the top white line of the ultrafiltration tube. Mix thoroughly and centrifuge until the upper layer volume is less than or equal to 100 μL (theoretical concentration of the anti-PD-1 antibody protein solution is ≥10 mg / mL). After centrifugation, the upper protein solution is aspirated and PBS is added to 100 μL (to a theoretical concentration of 10 mg / mL) to obtain SMCC-Keytruda. Centrifugation parameters: centrifugal force 3800 g, acceleration speed 8, deceleration speed 8, temperature 4°C.

[0589] Analysis of the coupling sites of SMCC-Keytruda

[0590] After the reaction, 200 μg of the SMCC-Keytruda sample was added to a solution containing 50 mmol / L Tris-HCl, 6 mol / L guanidine hydrochloride, and 20 mmol / L DTT, and incubated at 37°C for 30 min. IAM was then added to a final concentration of 50 mmol / L, and the solution was incubated at room temperature in the dark for 30 min to protect sulfhydryl groups. The solution was then replaced with enzyme cleavage buffer (50 mmol / L Tris-HCl, 0.8 mol / L urea, and 5 mmol / L EDTA) to a volume of 500 μL. The solution was centrifuged at 13,000 rpm for 15 min. The sample was recovered and made up to 500 μL with enzyme cleavage buffer. 100 μL of the sample was then added with Trypsin at a protein:enzyme ratio of 20:1 (mass ratio) and incubated at 37°C for 0.5 h. The reaction was terminated by adding formic acid to a final concentration of 1%. The treated sample was analyzed by LC-MS / MS.

[0591] The sample-peptide UV spectrum is shown in Figure 14.

[0592] SK sample-MCC junction site:

[0593] Example 10. SMCC-BJ (Tislelizumab) peptide mapping and coupling site analysis

[0594] Preparation of SMCC-Tislelizumab

[0595] Take 1mg / ml sulfo-SMCC solution, draw 21.8μL and add it to 138.2μL PBS buffer, and mix it evenly by blowing gently; draw 40μL anti-PD-1 antibody solution ( Add the sulfo-SMCC reagent (Baijen Bio) to the EP tube and mix thoroughly to a final concentration of 0.25 mM. Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 hour to obtain the treated anti-PD-1 antibody protein solution.

[0596] After the reaction is complete, the upper layer of the ultrafiltration tube is aspirated, and the treated anti-PD-1 antibody protein solution is added. PBS buffer is added to the top white line of the ultrafiltration tube and centrifuged. After centrifugation, the upper ultrafiltration tube is removed, the waste liquid in the lower tube is poured out, the upper ultrafiltration tube is replaced, and PBS buffer is added to the top white line of the ultrafiltration tube. Mix thoroughly and centrifuge until the upper layer volume is less than or equal to 100 μL (theoretical concentration of the anti-PD-1 antibody protein solution is ≥10 mg / mL). After centrifugation, the upper protein solution is aspirated and PBS is added to 100 μL (to a theoretical concentration of 10 mg / mL) to obtain SMCC-Tislelizumab. Centrifugation parameters: centrifugal force 3800 g, acceleration speed 8, deceleration speed 8, temperature 4°C.

[0597] The different batches of SMCC-BJ are shown in the following table.

[0598] The sample-peptide UV spectra are shown in Figures 15A and 15B.

[0599] S-BJ sample-MCC junction site:

[0600] Example 11: Different Length SMCCs (Different Linkers) Treating Keytruda (mRBC)

[0601] Coupling experiments with SMCCs of varying lengths were performed according to Example 3, with only the linker being different. Experimental information is shown in the following table and Figure 16 .

[0602] The MFI data show that NHS-PEG2-Mal and NHS-PEG24-Mal exhibit roughly the same coupling efficiency with SMCC. Both can be coupled to Keytruda via the aforementioned process, achieving 100% binding to red blood cells. Drug loading trends are consistent with the MFI values, with NHS-PEG24-Mal exhibiting slightly lower drug loading.

[0603] Example 12: SMCC-GGG peptide coupled to erythrocytes, extraction of erythrocyte membrane proteins, and mass spectrometry detection of protein types and amino acid types modified by SMCC-GGG on membrane proteins

[0604] 12.1 Preparation of Engineered Red Blood Cells (RBC-SMCC-GGG)

[0605] Engineered linker-ligated erythrocytes were prepared as described in Examples 1.1-1.3, except that Sulfo-SMCC-GGG was used instead of Sulfo-SMCC.

[0606] 12.2 RBC membrane protein extraction

[0607] Membrane collection

[0608] Take the preparation sample in 12.1, centrifuge at 1000g for 5 minutes, and remove the supernatant. Add isotonic PB solution (phosphate buffer, 100mM, pH7.4) to the precipitate, resuspend it, and centrifuge at 1000g for 5 minutes to remove the supernatant. Add hypotonic PB solution (10mM, pH7.4) to the washed RBCs and mix until the solution turns transparent and dark red. After standing at 4℃ for 30 minutes, centrifuge at 10000g for 10 minutes at 4℃. Discard the dark red supernatant, resuspend the red flocculent precipitate in hypotonic PB solution, and centrifuge at 10000g for 10 minutes at 4℃. Repeat the washing process 3-4 times until a milky white precipitate appears and is collected as membrane protein.

[0609] SDS-Page separation and Western blot identification

[0610] After non-reducing denaturation, membrane proteins were separated by electrophoresis using 8%-20% Page 1 gel. After transfer to PVDF membrane, the membranes were incubated with UOX antibody and HRP-labeled secondary antibodies, respectively. After TMB staining, the SDS-Page bands were identified as 60-80 kD, 100-150 kD, and >150 kD based on Western blotting results.

[0611] In-gel enzymatic digestion

[0612] The sample load was increased to 50 μg. After SDS-Page separation, the three bands were excised, decolorized, washed, and dried. Dithiothreitol was added for denaturation, iodoacetamide was blocked, and trypsin was used to terminate the enzymatic reaction. The supernatant was extracted and analyzed by mass spectrometry.

[0613] 12.3 Mass spectrometry detection

[0614] The peptide of 1452.55 Da ( FIG. 17 ) was used as the modification of the SMCC-GGG peptide, and the names of proteins containing each modified peptide were detected by comparison with the human protein database.

[0615] The detection results ( FIG17B ) showed that Band 3 protein, a protein associated with the erythrocyte membrane, was detected to contain the SMCC-GGG peptide modification.

[0616] Example 13: Peptide mapping of SMCC-ZJM and SMCC-TSQ, and analysis of coupling sites

[0617] Using the method as in Example 1, Sugemalimab (ZJM) and Atezolizumab (TSQ) were modified with SMCC to obtain SMCC-ZJM and SMCC-TSQ, respectively.

[0618] Peptide mapping sample processing

[0619] A 200 μg sample was added to a solution containing 50 mmol / L Tris-HCl, 6 mol / L guanidine hydrochloride, and 20 mmol / L DTT, and incubated at 60°C for 30 min. IAM was then added to a final concentration of 50 mmol / L, and the solution was incubated at room temperature in the dark for 30 min to protect sulfhydryl groups. The solution was then replaced with enzyme cleavage buffer (50 mmol / L Tris-HCl, 0.8 mol / L urea, and 5 mmol / L EDTA) to a volume of 500 μL. The solution was centrifuged at 13,000 rpm for 15 min, and the sample was recovered and filled to 500 μL with enzyme cleavage buffer. 100 μL of this volume was then added to a solution containing trypsin and LysC at a protein:enzyme ratio of 20:1 (mass ratio), and the solution was incubated at 37°C for 4 h. The reaction was terminated by adding formic acid to a final concentration of 1%. The treated sample was analyzed by LC-MS / MS.

[0620] The UV spectrum of the SMCC-ZJM&TSQ sample-peptide map is shown in Figure 18.

[0621] SMCC-ZJM sample-MCC junction site

[0622] SMCC-TSQ sample-MCC junction site

[0623] Example 14. Preparation of red blood cells targeting PD-L1 (taking the reaction of 1 mL of concentrated RBC as an example)

[0624] 14.1 The reagents and consumables used are shown in the following table:

[0625] 14.2 Preparation of Engineered Red Blood Cells

[0626] Acquisition of red blood cells

[0627] Open the cap of the blood collection tube and use a disposable syringe to transfer the blood sample from the tube to a blood bag. Once completed, seal the tube twice with a sterile tube sealer. Use a leukocyte filter to remove leukocytes from the whole blood. Using a 20mL disposable syringe, transfer the leukocyte-depleted blood sample from the blood storage bag to a 50mL centrifuge tube and centrifuge to collect red blood cells. Centrifuge to obtain a concentrated red blood cell intermediate. Use a disposable sterile pipette or a 1000μL pipette with the corresponding tip to discard the supernatant. Use an electric pipette to aspirate the concentrated red blood cells for subsequent use.

[0628] TCEP-treated red blood cells

[0629] After obtaining red blood cells, add 1× PBS, then TCEP solution, in a suitable reaction vessel, in a specific ratio. Mix thoroughly before adding concentrated red blood cells to a final TCEP concentration of 5 mM and a red blood cell concentration of 5e9 / mL. Place the 5mL centrifuge tube in a rotator and rotate for 60 minutes. After the reaction is complete, centrifuge the reaction solution, remove the supernatant, and wash twice with 10 volumes of 1× PBS by centrifugation to obtain the TCEP-treated concentrated RBCs.

[0630] Preparation of SMCC reaction solution

[0631] Take out sulfo-SMCC powder from the -20°C freezer, accurately weigh 2 mg and place it in an EP tube, add 1.8 ml of sterile water, shake to dissolve it completely, and then add 200 μL of 10X PBS to adjust the osmotic pressure. The concentration after dissolution is 1 mg / ml.

[0632] αPD-L1 protein processing

[0633] Take 218 μL of the 1 mg / mL SMCC solution and add it to 449 μL of PBS buffer. Gently pipette to mix thoroughly. Pipette 5 mg of the αPD-L1 antibody solution into an EP tube and mix thoroughly to a final SMCC concentration of 0.125 mM. Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 hour. After the reaction is complete, aspirate the upper layer of the ultrafiltration tube, add the treated αPD-L1 protein solution, add PBS buffer to the top white line of the ultrafiltration tube, and centrifuge. After centrifugation, remove the upper ultrafiltration tube, pour out the waste liquid from the lower tube, replace the upper ultrafiltration tube, add PBS buffer to the top white line of the ultrafiltration tube, mix thoroughly, and centrifuge. Centrifuge until the upper layer volume is ≤ 500 μL (theoretical αPD-L1 protein concentration ≥ 10 mg / mL). After centrifugation, aspirate the upper layer of the αPD-L1 protein solution, determine the protein concentration, and set aside.

[0634] Erythrocytes are conjugated to αPD-L1 antibodies to generate engineered erythrocytes

[0635] Add 1000 μL of modified concentrated RBCs to the ultrafiltered αPD-L1 protein solution and gently pipette to mix thoroughly. The final αPD-L1 concentration is 5 mg / mL and the RBC concentration is 5e9 / mL. Place the EP tube on a rotary mixer at 10 rpm and 30°C for 1 hour. After the reaction is complete, centrifuge and remove the supernatant. Add PBS (9 times the volume of concentrated RBCs), mix thoroughly, centrifuge, and remove the supernatant. Repeat this step twice.

[0636] Storage of engineered red blood cells

[0637] Pipette 1000 μL of saline and 500 μL of red blood cell preservation solution (saline equal to the volume of concentrated red blood cells and 0.5 times the volume of red blood cell preservation solution), add the engineered red blood cells, and gently pipette to mix thoroughly to obtain the engineered red blood cell preparation solution. Add the engineered red blood cell preparation solution to the packaging container. After packaging, store the preparation at 4°C until ready for use.

[0638] 14.3 Evaluation of Drug Loading in Engineered Red Blood Cell Preparations

[0639] Detection method of drug loading of RBCs-αPD-L1

[0640] First, a 5μL sample of red blood cell preparation was diluted 1000-fold and the red blood cells were counted using a cell counter. Then, 5μL of the red blood cell preparation was added to 95μL of RIPA solution to obtain the red blood cell preparation lysate. The drug loading of the red blood cell lysate was determined by enzyme-linked immunosorbent assay (ELISA). The final drug loading was calculated by converting the number of red blood cells contained in 5μL of the red blood cell preparation into the measured drug loading.

[0641] The results are shown in Figure 20. The data summary shows that the distribution range of RBCs-αPD-L1 drug loading is 200 μg / 10 10 cells or so.

[0642] Example 15. Pharmacokinetic evaluation of RBCs-αPD-L1 in C57BL / 6 mice

[0643] 15.1 Study Objectives

[0644] The purpose of this example is to evaluate the pharmacokinetics of mouse engineered red blood cells coupled to αPD-L1-1, αPD-L1-2, αPD-L1-3 and αPD-L1-4 (RBCs-αPD-L1-1, RBCs-αPD-L1-2, RBCs-αPD-L1-3 and RBCs-αPD-L1-4) prepared by Example 14 in C57BL / 6 mice.

[0645] 15.2 Research Methods

[0646] The prepared mouse engineered erythrocytes coupled to αPD-L1 were cultured at a rate of 1×10 9The cells were injected into C57BL / 6 mice via the tail vein. In this experiment, each type of engineered red blood cells was injected into 3 mice, for a total of 12 mice. The injections were performed every 3 days. The day of injection was recorded as day 0, and peripheral blood was collected from the mice on days 3, 6, 7, and 15. The proportion of RBCs-αPD-L1 positive cells in the peripheral blood of the mice was analyzed by flow cytometry (using PE anti-Human IgG Fc antibody, Biolegend, 409304) to detect the retention of RBCs-αPD-L1 in the body at different time points.

[0647] 15.3 Experimental Results

[0648] All data are expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism 9.

[0649] The results are shown in Figure 21: After three injections of mouse engineered red blood cells coupled to αPDL1-1 antibody (RBCs-αPD-L1-1), the ratio D0-D16 in the mouse body remained stable; after three injections of mouse engineered red blood cells coupled to αPDL1-3 antibody (RBCs-αPD-L1-3), the ratio D0-D16 in the mouse body remained stable; after three injections of mouse engineered red blood cells coupled to αPDL1-4 antibody (RBCs-αPD-L1-4), the ratio D0-D16 in the mouse body remained stable. The pharmacokinetic analysis showed that the pharmacokinetic index remained stable from D0 to D16, but was inferior to that of the mouse engineered red blood cell-coupled αPDL1-1 antibody (RBCs-αPD-L1-1) and the mouse engineered red blood cell-coupled αPDL1-3 antibody (RBCs-αPD-L1-3). The proportion of the mouse engineered red blood cell-coupled αPDL1-2 antibody (RBCs-αPD-L1-2) in the mice decreased by approximately 60% on D3 after the first injection, and the pharmacokinetic index decreased significantly after the next two injections, falling below the detection limit.

[0650] 15.4 Experimental Conclusion

[0651] Under the above process conditions, the engineered red blood cell-coupled PD-L1 antibody has good pharmacokinetic properties in mice, with the stability ratio being: RBCs-PD-L1-1 = RBCs-PD-L1-3 > RBCs-PD-L1-2 > RBCs-PD-L1-4.

[0652] 16. Pharmacodynamic evaluation of RBCs-αPD-L1 in an αPD-L1 antibody-responsive tumor model (hPD-L1+MC38)

[0653] 16.1 Study Objectives

[0654] The purpose of this study was to evaluate the effect of mouse engineered erythrocytes coupled to αPD-L1-1 and αPD-L1-4 on hPD-L1 in PD-L1 humanized mice (Jicui Yaokang). + Tumor killing function in MC38 cell-bearing tumor model.

[0655] 16.2 Research Methods

[0656] Mouse colon cancer cells hPD-L1 + MC38 was injected into mice subcutaneously at a rate of 1×10^6 cells until the tumor size of the mice reached about 64 mm. 3 At the same time, 32 experimental mice were randomly divided into 4 groups (n=8 / group): (1) control group, blank RBCs (1e9 cells / time, Q3D, for 4 times); (2) αPD-L1-1 treatment group (10 mg / kg / time, Q3D, for 4 times); (3) RBCs-αPD-L1-1 treatment group (1e9 cells / time, Q3D, for 4 times, with an average dose of about 1.2 mg / kg each time); (4) RBCs-αPD-L1-4 treatment group (1e9 cells / time, Q3D, for 4 times, with an average dose of about 1.2 mg / kg each time). The tumor volume was measured every 2-3 days. When the tumor in the control group was about 500 mm 3 The mice were killed at 4 pm and the tumor volumes of the mice in the four groups were compared.

[0657] The dosage of red blood cells coupled to the antibody is calculated as follows:

[0658] Red blood cell loading (mg anti-PD-L1 antibody / e10 RBCs)*number of cells administered (e10 / kg) = 1.2 mg / kg

[0659] 16.3 Research Results

[0660] All data are expressed as mean ± SEM and were analyzed using GraphPad Prism 9. Comparisons between two groups were performed using the t-test. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0661] The results are shown in Figure 22. Statistical analysis of tumor volume data during the efficacy phase revealed that, compared with the control group (G1, Control RBCs), the αPD-L1-1 treatment group (G2, 10 mg / kg) showed significant tumor growth inhibition starting on Day 3 after administration, reaching a peak tumor inhibition rate of 65.57% on Day 11, with a statistically significant difference (***P < 0.001). The RBCs-αPD-L1-1 treatment group (G3) showed significant tumor growth inhibition starting on Day 3 after administration, reaching a peak tumor inhibition rate of 49.73% on Day 11, with a statistically significant difference (***P < 0.001). The RBCs-αPD-L1-4 treatment group (G4) showed significant tumor growth inhibition starting on Day 3 after administration, reaching a peak tumor inhibition rate of 57.45% on Day 11, with a statistically significant difference (***P < 0.001). In summary, the mouse engineered red blood cells coupled with αPD-L1-1 or αPD-L1-4 administration groups had a significant effect on hPD-L1 + It has a significant tumor-suppressing effect on MC38 mouse colon cancer cell line-bearing mice.

[0662] 16.4 Experimental Conclusion

[0663] Effect of αPD-L1-1 or αPD-L1-4 on hPD-L1 in mice + It has a significant tumor-suppressing effect on MC38 mouse colon cancer cell line-bearing mice.

[0664] 17. RBCs-αPD-L1 (RBCs-αPD-L1-1 and RBCs-αPD-L1-3) in tumor models that do not respond to αPD-L1 antibodies (hPD-L1 - Pharmacodynamic evaluation in MC38)

[0665] 17.1 Study Objectives

[0666] The purpose of this study was to evaluate the effects of mouse engineered erythrocytes coupled to αPD-L1-1 and αPD-L1-3 on hPD-L1 in PD-L1 humanized mice (Jicui Yaokang). - Tumor killing function in MC38 cell-bearing tumor model.

[0667] 17.2 Research Methods

[0668] hPD-L1 in mouse colon cancer - MC38 cells were injected subcutaneously into mice at a cell count of 1×10^5. 3At the same time, 20 experimental mice were randomly divided into 4 groups (n=5 / group): (1) control group, blank RBCs (1e9 cells / time, twice a week, a total of 5 times); (2) αPD-L1-3 treatment group (10 mg / kg / time, twice a week, a total of 5 times); (3) RBCs-αPD-L1-1 treatment group (1e9 cells / time, twice a week, a total of 5 times, an average of about 0.4 mpk each time.); (4) RBCs-αPD-L1-3 treatment group (1e9 cells / time, twice a week, a total of 5 times, an average of about 0.4 mpk each time.). The tumor size was measured every 2-3 days. When the tumor in the control group was about 500 mm 3 The mice were killed at 4 pm and the tumor volumes of the mice in the four groups were compared.

[0669] 17.3 Experimental Results

[0670] All data are expressed as mean ± SEM and statistically analyzed using GraphPad Prism 9. Comparisons between two groups were performed using t-test. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001

[0671] The results are shown in Figure 23. According to the statistical analysis of tumor volume data during the efficacy process, both the RBCs-αPD-L1-1 treatment group (G3) and the RBCs-αPD-L1-3 treatment group (G4) showed a certain tumor inhibition effect. Among them, the RBCs-αPD-L1-1 treatment group (G3) showed obvious tumor growth inhibition from D7 after administration, and reached the highest peak of tumor inhibition rate on D14 (TGItv = 80.2%), and showed a statistically significant difference (*P < 0.05); the RBCs-αPD-L1-3 treatment group (G4) also showed a certain tumor inhibition effect (TGItv = 30.8%).

[0672] 17.4 Experimental Conclusion

[0673] Effect of αPD-L1-1 on hPD-L1 in mice engineered erythrocytes - It has a significant tumor-suppressing effect on MC38 mouse colon cancer cell line-bearing mice.

[0674] 18. RBCs-αPD-L1 (RBCs-αPD-L1-1 and RBCs-αPD-L1-4) in tumor models that do not respond to αPD-L1 antibodies (hPD-L1 - Pharmacodynamic evaluation of MC38)

[0675] 18.1 Study Objectives

[0676] The purpose of this study was to evaluate the effects of mouse engineered erythrocytes coupled to αPD-L1-1 and αPD-L1-4 on hPD-L1 in PD-L1 humanized mice (Jicui Yaokang). - Tumor killing function in MC38 cell-bearing tumor model.

[0677] 18.2 Research Methods

[0678] hPD-L1 in mouse colon cancer - MC38 cells were injected subcutaneously into mice at a cell count of 2.5×10^5. The mice were then treated with a 22mm tumor. 3 At the time of the experiment, 24 experimental animals were randomly divided into 4 groups (n=6 / group): (1) control group, blank RBCs (1e9 cells / time, twice a week, a total of 5 times); (2) αPD-L1-1 treatment group (10 mg / kg / time, twice a week, a total of 5 times); (3) RBCs-αPD-L1-1 treatment group (1e9 cells / time, twice a week, a total of 5 times, an average of about 0.5 mpk each time); (4) RBCs-αPD-L1-4 treatment group (1e9 cells / time, twice a week, a total of 5 times, an average of about 0.5 mpk each time). The tumor size was measured every 2-3 days. When the tumor in the control group was about 800 mm 3 The mice were killed at 4 pm and the tumor volumes of the mice in the four groups were compared.

[0679] 18.3 Experimental Results

[0680] All data are expressed as mean ± SEM and statistically analyzed using GraphPad Prism 9. Comparisons between two groups were performed using t-test. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001

[0681] The results are shown in Figure 24. According to the statistical analysis of tumor volume data during the efficacy process, compared with the control group (G1 (Control RBCs)): the αPD-L1-1 treatment group (G2, 10 mg / kg) did not show significant tumor growth inhibition; the RBCs-αPD-L1-1 treatment group (G3) showed significant tumor growth inhibition starting from D5 after administration, reaching the highest peak tumor inhibition rate on D10 (TGItv = 60.3%), and showing a statistically significant difference (*P < 0.05); the RBCs-αPD-L1-4 treatment group (G4) showed significant tumor growth inhibition starting from D5 after administration, reaching the highest peak tumor inhibition rate on D10 (TGItv = 67.9%), and showing a statistically significant difference (**P < 0.01).

[0682] 18.4 Experimental Conclusion

[0683] Effect of αPD-L1-1 / αPD-L1-4 on hPD-L1 in mouse engineered erythrocytes - It has a significant tumor-suppressing effect on MC38 mouse colon cancer cell line-bearing mice.

[0684] Sequence Listing

Claims

1. An antibody-erythrocyte conjugate comprising an erythrocyte conjugated to an antibody or an antigen-binding fragment thereof, in, The antibody or its antigen-binding fragment is coupled to a membrane protein on red blood cells, such as band 3 protein, via a linker. Preferably, the antibody or antigen-binding fragment thereof is a PD-L1 antibody or an antigen-binding fragment thereof, and the red blood cell is a mature red blood cell such as an adult natural red blood cell.

2. The antibody-erythrocyte conjugate of claim 1, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain variable regions CDR1, CDR2 and CDR3, and light chain variable regions CDR1, CDR2 and CDR3, wherein the heavy chain variable regions CDR1, CDR2 and CDR3, and light chain variable regions CDR1, CDR2 and CDR3 are from the antibodies disclosed in CN102245640A or CN106432501A or CN102918058A, or sugemalimab or atezolizumab or durvalumab; or The PD-L1 antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3 of VHH, wherein CDR1, CDR2 and CDR3 of VHH are from any antibody disclosed in CN107849130A or Envolizumab.

3. The antibody-erythrocyte conjugate of claim 1, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region are from the antibodies disclosed in CN102245640A or CN106432501A or CN102918058A, or sugemalimab or atezolizumab or durvalumab; or The PD-L1 antibody or antigen-binding fragment thereof comprises VHH, wherein the VHH is derived from any antibody disclosed in CN107849130A or Envolizumab.

4. The antibody-erythrocyte conjugate of claim 1, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain and the light chain are from the antibodies disclosed in CN102245640A or CN106432501A or CN107849130A or CN102918058A, or sugemalimab or atezolizumab or durvalumab; or The PD-L1 antibody or antigen-binding fragment thereof comprises a fusion antibody of VHH and Fc, for example, any antibody disclosed in CN107849130A or Envolizumab.

5. The antibody-erythrocyte conjugate of claim 1, wherein the PD-L1 antibody or its antigen-binding fragment is selected from the antibodies disclosed in CN102245640A or CN106432501A or CN107849130A or CN102918058A, or is selected from sugemalimab or atezolizumab or durvalumab or envolimab or its antigen-binding fragment.

6. The antibody-erythrocyte conjugate of claim 1, wherein the PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain variable regions CDR1 (HCDR1), CDR2 (HCDR2) and CDR3 (HCDR3), and light chain variable regions CDR1 (LCDR1), CDR2 (LCDR2) and CDR3 (LCDR3), wherein (i) the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 8, the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:10; and the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:3, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:4, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:5; (ii) the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 19, CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:20; and the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO:13, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:14, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:15; or (iii) the HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 31, CDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 32; and the LCDR1 comprises SEQ ID The amino acid sequence shown in SEQ ID NO:25 or consists of it, LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO:26, and LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO:

27.

7. The antibody-erythrocyte conjugate of claim 1, wherein the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, or consists of the amino acid sequence, and The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 2, or consists of the amino acid sequence; (ii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, or consists of the amino acid sequence, and The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 12, or consists of the amino acid sequence; or (iii) the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29, or consists of the amino acid sequence, and The light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 24, or consists of the amino acid sequence.

8. The antibody-erythrocyte conjugate of claim 1, wherein The anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein (i) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 6, or consists of the amino acid sequence, and The light chain comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the amino acid sequence; (ii) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 16, or consists of the amino acid sequence, and The light chain comprises the amino acid sequence shown in SEQ ID NO: 1, or consists of the amino acid sequence; or (iii) the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 28, or consists of the amino acid sequence, and The light chain comprises the amino acid sequence shown in SEQ ID NO: 23, or consists of the amino acid sequence; Alternatively, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a fusion antibody of VHH and Fc, which comprises the amino acid sequence shown in SEQ ID NO: 22, or consists of the amino acid sequence.

9. The antibody-erythrocyte conjugate of any one of claims 1 to 8, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), bivalent antibody or fragment thereof, or linear antibody or half antibody.

10. The antibody-erythrocyte conjugate of any one of claims 1-8, wherein the linker has a functional group that can react with a thiol group on a cysteine ​​residue present on an RBC membrane protein to form a covalent bond to the RBC, and has a functional group that can react with an amino group (-NH2) on a lysine in the antibody structure to connect to the antibody.

11. The antibody-erythrocyte conjugate of any one of claims 1 to 3, wherein the linker is LC-SMCC, SMCC-GGG, SMCC or Sulfo-SMCC or NHS-PEGn-MAL.

12. The antibody-erythrocyte conjugate of any one of claims 1 to 8, wherein the erythrocytes are obtained by a method comprising: (i) separating and concentrating red blood cells from human whole blood, optionally by filtering out leukocytes; (ii) treating red blood cells with a thiol reducing agent to chemically modify the surface of red blood cells; (iii) The modified red blood cells are collected and concentrated.

13. The antibody-erythrocyte conjugate of claim 12, wherein the thiol reducing agent is TCEP.

14. A method for preparing the antibody-erythrocyte conjugate according to any one of claims 1 to 11, comprising (1) reacting a nucleophilic group of the antibody or antigen-binding fragment thereof with a bifunctional linker reagent to form an antibody or antigen-binding fragment thereof connected to the linker via a covalent bond; Antigen binding fragment, (2) chemically modifying the surface of adult natural red blood cells to expose nucleophilic groups on the red blood cells; (3) mixing the antibody or antigen-binding fragment thereof connected to a linker in (1) and the red blood cells obtained in (2) such that the two are covalently coupled via the linker; (4) Collecting the antibody-erythrocyte conjugate obtained in (3).

15. The method of claim 14, wherein the nucleophilic group on the antibody or antigen-binding fragment thereof is an N-terminal amine group or a side chain amine group, and the nucleophilic group on the red blood cell is a side chain thiol group.

16. The method of claim 15, wherein the side chain thiol group is a thiol group after reduction of cysteine, for example, the cysteine ​​is from Band3, for example, from the fragment shown in SEQ ID NO:21 of Band3.

17. The method of claim 15 or 16, wherein the red blood cells are chemically modified using a thiol reducing agent.

18. The method of claim 17, wherein the thiol reducing agent is TCEP.

19. The method of claim 18, wherein the final concentration of TCEP is between 0.1 mM and 50 mM, such as 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 5.0 mM.

20. A pharmaceutical composition or preparation comprising the antibody-erythrocyte conjugate of any one of claims 1 to 11, or the antibody-erythrocyte conjugate prepared by the method of any one of claims 12 to 19, and a pharmaceutical excipient.

21. The pharmaceutical composition or formulation of claim 20, wherein the formulation is a human blood preparation, such as an autologous or allogeneic human blood preparation.

22. The pharmaceutical composition or formulation of claim 21, wherein the human blood preparation is a human leukopenic blood preparation.

23. The pharmaceutical composition or formulation of claim 21 or 22, wherein the human blood preparation comprises 10-1000 μg / mL of the antibody-erythrocyte conjugate, for example, above 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL of the antibody-erythrocyte conjugate.

24. Use of the antibody-erythrocyte conjugate of any one of claims 1 to 11, or the antibody-erythrocyte conjugate prepared by the method of any one of claims 12 to 19, in the preparation of a medicament or a human blood preparation for stimulating the host's immune system (e.g., enhancing the cellular immune response) or treating cancer in a subject.

25. The use of claim 24, wherein the cancer is a solid tumor.

26. The use of claim 24 or 25, wherein the cancer is resistant or insensitive to anti-immune checkpoint blockade therapy.

27. The use according to claim 24 or 25, wherein the cancer is a cancer that is resistant or insensitive to anti-PD-1 antibody or anti-PD-L1 antibody.

28. () The use according to any one of claims 24 to 27, wherein the red blood cells in the antibody-red blood cell conjugate are autologous red blood cells or allogeneic red blood cells.

29. The use according to any one of claims 24 to 28, wherein the medicament or human blood preparation is administered in combination with one or more therapies, such as treatment modalities or other therapeutic agents.

30. The use of claim 29, wherein the therapy is immunotherapy, or the treatment modality is radiotherapy or surgical treatment, and / or the therapeutic agent is selected from chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, etc., preferably, the therapeutic agent is selected from tumor vaccines, immune checkpoint inhibitor antibodies or immune agonist antibodies.

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