Anti-rhesus D human monoclonal antibody

Abstract

The present invention provides recombinant antibodies that bind to RhD blood group antigens as well as compositions comprising these recombinant antibodies and methods for selecting combinations of different recombinant antibodies derived from donor blood. The recombinant antibodies are useful in the prevention of neonatal hemolytic disease (HDN), the treatment of idiopathic thrombocytopenic purpura (ITP), and the prevention of anti-RhD alloimmunity in RhD negative patients receiving RhD + transfusion.

Description

[0001] Related applications This application claims priority to Australian provisional patent application AU2023902692, filed on 23 August 2023, the entire contents of which are incorporated herein by reference.

[0002] References to sequence lists The entire contents of the sequence list submitted electronically are incorporated by reference for all purposes. Technical Field

[0003] This invention generally relates to recombinant antibodies that bind to RhD blood group antigens. These antibodies can be used to treat and prevent hemolytic disease of the newborn (HDN), treat idiopathic thrombocytopenic purpura (ITP), and prevent the absorption of RhD. + Anti-RhD allogeneic immunity in RhD-negative patients receiving blood transfusions. Background Technology

[0004] Antibodies are naturally occurring molecules produced by B lymphocytes of the immune system that help fight infection and disease. Rhesus D (RhD) is a blood type found on human red blood cells. Approximately 85% of people are RhD positive (RhD...). + 15% of people are RhD negative (RhD - However, these frequencies vary across different groups. When RhD - Mother is pregnant with RhD + During infancy, fetal red blood cells (RBCs) enter the mother's circulation, sensitizing the mother and triggering an antibody response against RhD molecules. This "anti-D response" intensifies with each pregnancy and can lead to the destruction of the baby's red blood cells, a condition known as hemolytic disease of the newborn (HDN). HDN can cause miscarriage or stillbirth and has historically been a leading cause of infant mortality, and remains so in many parts of the world.

[0005] In the 1960s, it was suggested that RhD could be considered. - Women are injected with anti-RhD serum (i.e., serum containing anti-RhD antibodies) to rapidly remove small amounts of fetal RhD that have entered the maternal circulation. + The blood cells are converted to prevent the mother's immune system from being sensitized. This proved to be very successful, and since then, standard clinical care has shifted to prophylactic treatment of RhD with anti-RhD serum. - Women (at 28 and 34 weeks of gestation). Therefore, HND is now rare in developed countries that have implemented anti-RhD programs.

[0006] Anti-RhD plasma is derived from a small group of unpaid blood donors selected to possess anti-RhD antibodies. These donors then receive RhD treatment regularly. + RBC stimulation is used to stimulate the immune system and maintain high levels of anti-RhD antibodies. Some donors have donated blood more than 1,000 times for this purpose, but such services are not available in many parts of the world. Furthermore, voluntary blood donors may experience discomfort after repeated immunizations with "foreign" RBCs. In addition, the donation process is time-consuming and expensive, and carries a small but not negligible risk of transmitting infectious diseases. For example, in the UK, donor plasma has been sourced from North America for many years to minimize any risk of transmission of variant Creutzfeldt-Jakob disease.

[0007] Therefore, there is a need to develop alternative non-plasma-derived anti-RhD antibodies. Summary of the Invention

[0008] The inventors have unexpectedly developed an alternative source of anti-D antibodies that is independent of plasma and donors. These anti-D antibodies can be used, for example, as a prophylactic treatment and / or therapeutic for potential sensitization events in Rh-negative women who are pregnant or recently pregnant (e.g., up to 10 days after termination of pregnancy).

[0009] Therefore, in one aspect of this disclosure, an isolated or recombinant antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD) is provided, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising: a complementarity-determining region (CDR) H1 comprising a sequence as shown in any of the following: SEQ ID NO: 11, SEQ ID NO: 43, SEQ ID NO: 75, SEQ ID NO: 107, SEQ ID NO: 139, SEQ ID NO: 171, SEQ ID NO: 203, SEQ ID NO: 235, SEQ ID NO: 267, SEQ ID NO: 299, SEQ ID NO: 331, SEQ ID NO: 363, SEQ ID NO: 395, SEQ ID NO: 427, SEQ ID NO: 459, SEQ ID NO: 491; and a CDR H2 comprising a sequence as shown in any of the following: SEQ ID NO: 13, SEQ ID NO: 45, SEQ ID NO: 463, SEQ ID NO: 395, SEQ ID NO: 427, SEQ ID NO: 459, SEQ ID NO: 491; and a CDR H2 comprising a sequence as shown in any of the following: SEQ ID NO: 13, SEQ ID NO: 45, SEQ ID NO: 463, SEQ ID NO: 495, SEQ ID NO: 491; 77, SEQ ID NO:109, SEQ ID NO:141, SEQ ID NO:173, SEQ ID NO:205, SEQ ID NO:237, SEQ ID NO:269, SEQ ID NO:301, SEQ ID NO:333, SEQ ID NO:365, SEQ ID NO:397, SEQ ID NO:429, SEQ ID NO:461, SEQ ID NO:493; and CDR H3, comprising a sequence as shown in any of the following: SEQ ID NO:15, SEQ ID NO:47, SEQ ID NO:79, SEQ ID NO:111, SEQ ID NO:143, SEQ ID NO:175, SEQ ID NO:207, SEQ ID NO:239, SEQ ID NO:271, SEQ ID NO:303, SEQ ID NO:335, SEQ ID NO:367, SEQ ID NO: 399, SEQ ID NO: 431, SEQ ID NO: 463, SEQ ID NO: 495.

[0010] In one embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:11, a CDR H2 sequence as shown in any one of SEQ ID NO:13, and a CDR H3 sequence as shown in SEQ ID NO:15.

[0011] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:43, a CDR H2 sequence as shown in SEQ ID NO:45, and a CDR H3 sequence as shown in SEQ ID NO:47.

[0012] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:75, a CDR H2 sequence as shown in SEQ ID NO:77, and a CDR H3 sequence as shown in SEQ ID NO:79.

[0013] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises the CDR H1 sequence as shown in SEQ ID NO: 107, the CDR H2 sequence as shown in SEQ ID NO: 109, and the CDR H3 sequence as shown in SEQ ID NO: 111.

[0014] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO: 139, a CDR H2 sequence as shown in SEQ ID NO: 141, and a CDR H3 sequence as shown in SEQ ID NO: 143.

[0015] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:171, a CDR H2 sequence as shown in SEQ ID NO:173, and a CDR H3 sequence as shown in SEQ ID NO:175.

[0016] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:203, a CDR H2 sequence as shown in SEQ ID NO:205, and a CDR H3 sequence as shown in SEQ ID NO:207.

[0017] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:235, a CDR H2 sequence as shown in SEQ ID NO:237, and a CDR H3 sequence as shown in SEQ ID NO:239.

[0018] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:267, a CDR H2 sequence as shown in SEQ ID NO:269, and a CDR H3 sequence as shown in SEQ ID NO:271.

[0019] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:299, a CDR H2 sequence as shown in SEQ ID NO:301, and a CDR H3 sequence as shown in SEQ ID NO:303.

[0020] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:331, a CDR H2 sequence as shown in SEQ ID NO:333, and a CDR H3 sequence as shown in SEQ ID NO:335.

[0021] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:363, a CDR H2 sequence as shown in SEQ ID NO:365, and a CDR H3 sequence as shown in SEQ ID NO:367.

[0022] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:395, a CDR H2 sequence as shown in SEQ ID NO:397, and a CDR H3 sequence as shown in SEQ ID NO:399.

[0023] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:427, a CDR H2 sequence as shown in SEQ ID NO:429, and a CDR H3 sequence as shown in SEQ ID NO:431.

[0024] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:459, a CDR H2 sequence as shown in SEQ ID NO:461, and a CDR H3 sequence as shown in SEQ ID NO:463.

[0025] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a CDR H1 sequence as shown in SEQ ID NO:491, a CDR H2 sequence as shown in SEQ ID NO:493, and a CDR H3 sequence as shown in SEQ ID NO:495.

[0026] In one or a further embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises a light chain variable region (VL) comprising: a CDR L1 sequence as shown in any of the following: SEQ ID NO: 27, SEQ ID NO: 59, SEQ ID NO: 91, SEQ ID NO: 123, SEQ ID NO: 155, SEQ ID NO: 187, SEQ ID NO: 219, SEQ ID NO: 251, SEQ ID NO: 283, SEQ ID NO: 315, SEQ ID NO: 347, SEQ ID NO: 379, SEQ ID NO: 411, SEQ ID NO: 443, SEQ ID NO: 475, SEQ ID NO: 507; and a CDR L2 sequence as shown in any of the following: SEQ ID NO: 29, SEQ ID NO: 61, SEQ ID NO: 93, SEQ ID NO: 125, SEQ ID NO: 157, SEQ ID NO: 507. 189, SEQ ID NO: 221, SEQ ID NO: 253, SEQ ID NO: 285, SEQ ID NO: 317, SEQ ID NO: 349, SEQ ID NO: 381, SEQ ID NO: 413, SEQ ID NO: 445, SEQ ID NO: 477, SEQ ID NO: 509; and CDR L3 sequences as shown in any of the following: SEQ ID NO: 31, SEQ ID NO: 63, SEQ ID NO: 95, SEQ ID NO: 127, SEQ ID NO: 159, SEQ ID NO: 191, SEQ ID NO: 223, SEQ ID NO: 255, SEQ ID NO: 287, SEQ ID NO: 319, SEQ ID NO: 351, SEQ ID NO: 383, SEQ ID NO: 415, SEQ ID NO: 447, SEQ ID NO: 479, SEQ ID NO: 511.

[0027] In one embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 27, a CDR L2 sequence as shown in SEQ ID NO: 29, and a CDR L3 sequence as shown in SEQ ID NO: 31.

[0028] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 59, a CDR L2 sequence as shown in SEQ ID NO: 61, and a CDR L3 sequence as shown in SEQ ID NO: 63.

[0029] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 91, a CDR L2 sequence as shown in SEQ ID NO: 93, and a CDR L3 sequence as shown in SEQ ID NO: 95.

[0030] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 123, a CDR L2 sequence as shown in SEQ ID NO: 125, and a CDR L3 sequence as shown in SEQ ID NO: 127.

[0031] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 155, a CDR L2 sequence as shown in SEQ ID NO: 157, and a CDR L3 sequence as shown in SEQ ID NO: 159.

[0032] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 187, a CDR L2 sequence as shown in SEQ ID NO: 189, and a CDR L3 sequence as shown in SEQ ID NO: 191.

[0033] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 219, a CDR L2 sequence as shown in SEQ ID NO: 221, and a CDR L3 sequence as shown in SEQ ID NO: 223.

[0034] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 251, a CDR L2 sequence as shown in SEQ ID NO: 253, and a CDR L3 sequence as shown in SEQ ID NO: 255.

[0035] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 283, a CDR L2 sequence as shown in SEQ ID NO: 285, and a CDR L3 sequence as shown in SEQ ID NO: 287.

[0036] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 315, a CDR L2 sequence as shown in SEQ ID NO: 317, and a CDR L3 sequence as shown in SEQ ID NO: 319.

[0037] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 347, a CDR L2 sequence as shown in SEQ ID NO: 349, and a CDR L3 sequence as shown in SEQ ID NO: 351.

[0038] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 379, a CDR L2 sequence as shown in SEQ ID NO: 381, and a CDR L3 sequence as shown in SEQ ID NO: 383.

[0039] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 411, a CDR L2 sequence as shown in SEQ ID NO: 413, and a CDR L3 sequence as shown in SEQ ID NO: 415.

[0040] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 443, a CDR L2 sequence as shown in SEQ ID NO: 445, and a CDR L3 sequence as shown in SEQ ID NO: 447.

[0041] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 475, a CDR L2 sequence as shown in SEQ ID NO: 477, and a CDR L3 sequence as shown in SEQ ID NO: 479.

[0042] In another embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which includes: a CDR L1 sequence as shown in SEQ ID NO: 507, a CDR L2 sequence as shown in SEQ ID NO: 509, and a CDR L3 sequence as shown in SEQ ID NO: 511.

[0043] In one or a further embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VH, which comprises an amino acid sequence having at least 70% sequence identity with the sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 41, SEQ ID NO: 73, SEQ ID NO: 105, SEQ ID NO: 137, SEQ ID NO: 169, SEQ ID NO: 201, SEQ ID NO: 233, SEQ ID NO: 265, SEQ ID NO: 297, SEQ ID NO: 329, SEQ ID NO: 361, SEQ ID NO: 393, SEQ ID NO: 425, SEQ ID NO: 457, and SEQ ID NO: 489.

[0044] In one or a further embodiment of this disclosure, the antibody or antigen-binding fragment of this disclosure comprises VL, which comprises an amino acid sequence having at least 70% sequence identity with the sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 57, SEQ ID NO: 89, SEQ ID NO: 121, SEQ ID NO: 153, SEQ ID NO: 185, SEQ ID NO: 217, SEQ ID NO: 249, SEQ ID NO: 281, SEQ ID NO: 313, SEQ ID NO: 345, SEQ ID NO: 377, SEQ ID NO: 409, SEQ ID NO: 441, SEQ ID NO: 473, and SEQ ID NO: 505.

[0045] On the other hand, this disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD), wherein the antibody or antigen-binding fragment thereof comprises: a) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 11; CDR H2, comprising the sequence shown in SEQ ID NO: 13; and CDR H3, comprising the sequence shown in SEQ ID NO: 15; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 27; CDR L2, which contains the sequence shown in SEQ ID NO: 29; and CDR L3, which contains the sequence shown in SEQ ID NO: 31. b) The heavy chain variable region (VH) comprises: a complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 43; a CDR H2, comprising the sequence shown in SEQ ID NO: 45; and a CDR H3, comprising the sequence shown in SEQ ID NO: 47; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 59; CDR L2, which contains the sequence shown in SEQ ID NO: 61; and CDR L3, which contains the sequence shown in SEQ ID NO: 63. c) The heavy chain variable region (VH) comprises: a complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 75; a CDR H2, comprising the sequence shown in SEQ ID NO: 77; and a CDR H3, comprising the sequence shown in SEQ ID NO: 79; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 91; CDR L2, which contains the sequence shown in SEQ ID NO: 93; and CDR L3, which contains the sequence shown in SEQ ID NO: 95; d) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 107; CDR H2, comprising the sequence shown in SEQ ID NO: 109; and CDR H3, comprising the sequence shown in SEQ ID NO: 111; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 123; CDR L2, which contains the sequence shown in SEQ ID NO: 125; and CDR L3, which contains the sequence shown in SEQ ID NO: 127. e) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 139; CDR H2, comprising the sequence shown in SEQ ID NO: 141; and CDR H3, comprising the sequence shown in SEQ ID NO: 143; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 155; CDR L2, which contains the sequence shown in SEQ ID NO: 157; and CDR L3, which contains the sequence shown in SEQ ID NO: 159; f) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 171; CDR H2, comprising the sequence shown in SEQ ID NO: 173; and CDR H3, comprising the sequence shown in SEQ ID NO: 175; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 187; CDR L2, which contains the sequence shown in SEQ ID NO: 189; and CDR L3, which contains the sequence shown in SEQ ID NO: 191; g) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 203; CDR H2, comprising the sequence shown in SEQ ID NO: 205; and CDR H3, comprising the sequence shown in SEQ ID NO: 207; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 219; CDR L2, which contains the sequence shown in SEQ ID NO: 221; and CDR L3, which contains the sequence shown in SEQ ID NO: 223; h) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 235; CDR2, comprising the sequence shown in SEQ ID NO: 237; and CDR3, comprising the sequence shown in SEQ ID NO: 239; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 251; CDR L2, which contains the sequence shown in SEQ ID NO: 253; and CDR L3, which contains the sequence shown in SEQ ID NO: 255; i) The heavy chain variable region (VH) comprises: a complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 267; a CDR H2, comprising the sequence shown in SEQ ID NO: 269; and a CDR3, comprising the sequence shown in SEQ ID NO: 271; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 283; CDR L2, which contains the sequence shown in SEQ ID NO: 285; and CDR L3, which contains the sequence shown in SEQ ID NO: 287. j) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 299; CDR H2, comprising the sequence shown in SEQ ID NO: 301; and CDR H3, comprising the sequence shown in SEQ ID NO: 303; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 315; CDR L2, which contains the sequence shown in SEQ ID NO: 317; and CDR L3, which contains the sequence shown in SEQ ID NO: 319. k) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 331; CDR H2, comprising the sequence shown in SEQ ID NO: 333; and CDR H3, comprising the sequence shown in SEQ ID NO: 335; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 347; CDR L2, which contains the sequence shown in SEQ ID NO: 349; and CDR L3, which contains the sequence shown in SEQ ID NO: 351; l) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 3631; CDR H2, comprising the sequence shown in SEQ ID NO: 365; and CDR H3, comprising the sequence shown in SEQ ID NO: 367; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 379; CDR L2, which contains the sequence shown in SEQ ID NO: 381; and CDR L3, which contains the sequence shown in SEQ ID NO: 383. m) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 395; CDR H2, comprising the sequence shown in SEQ ID NO: 397; and CDR H3, comprising the sequence shown in SEQ ID NO: 399; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 411; CDR L2, which contains the sequence shown in SEQ ID NO: 413; and CDR L3, which contains the sequence shown in SEQ ID NO: 415. n) Heavy chain variable region (VH), comprising: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 427; CDR H2, comprising the sequence shown in SEQ ID NO: 429; and CDR H3, comprising the sequence shown in SEQ ID NO: 431; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 443; CDR L2, which contains the sequence shown in SEQ ID NO: 445; and CDR L3, which contains the sequence shown in SEQ ID NO: 447. o) Heavy chain variable region (VH), comprising: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 459; CDR H2, comprising the sequence shown in SEQ ID NO: 461; and CDR H3, comprising the sequence shown in SEQ ID NO: 463; and The light chain variable region (VL) comprises: CDR L1, containing the sequence shown in SEQ ID NO: 475; CDR L2, containing the sequence shown in SEQ ID NO: 477; and CDR L3, containing the sequence shown in SEQ ID NO: 479; or p) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 491; CDR H2, comprising the sequence shown in SEQ ID NO: 493; and CDR H3, comprising the sequence shown in SEQ ID NO: 495; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 507; CDR L2, which contains the sequence shown in SEQ ID NO: 509; and CDR L3, which contains the sequence shown in SEQ ID NO: 511.

[0046] On the other hand, this disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD), wherein the antibody or antigen-binding fragment thereof comprises: a) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 9, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 25; b) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 41 and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 57; c) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 73, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 89; d) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 105 and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 121; e) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 137, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 153; f) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 169, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 185. g) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 201, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 217. h) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 233, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 249; i) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 265, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 281; j) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 297, and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 313; k) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 329, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 345; l) a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 361, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 377; m) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 393, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 409; n) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 425, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 441. o) comprising a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 457, and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 473; or p) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 489, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 505.

[0047] In some embodiments of any of the foregoing aspects of this disclosure, the antibody or antigen-binding fragment is capable of specifically binding to RhD-positive (RhD+) RBCs but not to RhD-negative (RhD-) RBCs. Advantageously, the antibody or antigen-binding fragment is capable of agglutinating RhD+ erythrocytes but not RhD- erythrocytes.

[0048] In some embodiments or further embodiments of any of the foregoing aspects of this disclosure, the antibody or antigen-binding fragment is one of IgG subclass 1, IgG subclass 2, or IgG subclass 3.

[0049] In some embodiments or further embodiments of any of the foregoing aspects of this disclosure, the antibody or antigen-binding fragment is IgG subclass 1, IgG, or IgG subclass 3.

[0050] In some embodiments or further embodiments of any of the foregoing aspects of this disclosure, the antibody or antigen-binding fragment is IgG subclass 1.

[0051] In some embodiments or further embodiments of any of the foregoing aspects of this disclosure, the antibody or antigen-binding fragment comprises an Fc region or a portion thereof.

[0052] Advantageously, one or more of the antibodies or antigen-binding fragments disclosed herein can be used in combination to provide a composition. Therefore, this disclosure provides a composition comprising one or more antibodies or antigen-binding fragments as defined herein. In some embodiments, one or more of the antibodies or antigen-binding fragments disclosed herein can be used in combination with another antibody or antigen-binding fragment capable of specifically binding to rhesus monkey D (RhD), which may optionally be identified using the methods disclosed herein.

[0053] Therefore, in another aspect, this disclosure provides a composition comprising one or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein the one or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 11; CDR H2, comprising the sequence shown in SEQ ID NO: 13; and CDR H3, comprising the sequence shown in SEQ ID NO: 15; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 27; CDR L2, which contains the sequence shown in SEQ ID NO: 29; and CDR L3, which contains the sequence shown in SEQ ID NO: 31. b) The heavy chain variable region (VH) comprises: a complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 43; a CDR H2, comprising the sequence shown in SEQ ID NO: 45; and a CDR H3, comprising the sequence shown in SEQ ID NO: 47; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 59; CDR L2, which contains the sequence shown in SEQ ID NO: 61; and CDR L3, which contains the sequence shown in SEQ ID NO: 63. c) The heavy chain variable region (VH) comprises: a complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 75; a CDR H2, comprising the sequence shown in SEQ ID NO: 77; and a CDR H3, comprising the sequence shown in SEQ ID NO: 79; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 91; CDR L2, which contains the sequence shown in SEQ ID NO: 93; and CDR L3, which contains the sequence shown in SEQ ID NO: 95; d) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 107; CDR H2, comprising the sequence shown in SEQ ID NO: 109; and CDR H3, comprising the sequence shown in SEQ ID NO: 111; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 123; CDR L2, which contains the sequence shown in SEQ ID NO: 125; and CDR L3, which contains the sequence shown in SEQ ID NO: 127. e) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 139; CDR H2, comprising the sequence shown in SEQ ID NO: 141; and CDR H3, comprising the sequence shown in SEQ ID NO: 143; and The light chain variable region (VL) comprises: CDR1, which contains the sequence shown in SEQ ID NO: 155; CDR L2, which contains the sequence shown in SEQ ID NO: 157; and CDR L3, which contains the sequence shown in SEQ ID NO: 159; f) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 171; CDR H2, comprising the sequence shown in SEQ ID NO: 173; and CDR H3, comprising the sequence shown in SEQ ID NO: 175; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 187; CDR L2, which contains the sequence shown in SEQ ID NO: 189; and CDR L3, which contains the sequence shown in SEQ ID NO: 191; g) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 203; CDR H2, comprising the sequence shown in SEQ ID NO: 205; and CDR H3, comprising the sequence shown in SEQ ID NO: 207; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 219; CDR L2, which contains the sequence shown in SEQ ID NO: 221; and CDR L3, which contains the sequence shown in SEQ ID NO: 223; h) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 235; CDR2, comprising the sequence shown in SEQ ID NO: 237; and CDR3, comprising the sequence shown in SEQ ID NO: 239; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 251; CDR L2, which contains the sequence shown in SEQ ID NO: 253; and CDR L3, which contains the sequence shown in SEQ ID NO: 255; i) The heavy chain variable region (VH) comprises: a complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 267; a CDR H2, comprising the sequence shown in SEQ ID NO: 269; and a CDR3, comprising the sequence shown in SEQ ID NO: 271; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 283; CDR L2, which contains the sequence shown in SEQ ID NO: 285; and CDR L3, which contains the sequence shown in SEQ ID NO: 287. j) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 299; CDR H2, comprising the sequence shown in SEQ ID NO: 301; and CDR H3, comprising the sequence shown in SEQ ID NO: 303; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 315; CDR L2, which contains the sequence shown in SEQ ID NO: 317; and CDR L3, which contains the sequence shown in SEQ ID NO: 319. k) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 331; CDR H2, comprising the sequence shown in SEQ ID NO: 333; and CDR H3, comprising the sequence shown in SEQ ID NO: 335; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 347; CDR L2, which contains the sequence shown in SEQ ID NO: 349; and CDR L3, which contains the sequence shown in SEQ ID NO: 351; l) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 363; CDR H2, comprising the sequence shown in SEQ ID NO: 365; and CDR H3, comprising the sequence shown in SEQ ID NO: 367; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 379; CDR L2, which contains the sequence shown in SEQ ID NO: 381; and CDR L3, which contains the sequence shown in SEQ ID NO: 383. m) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 395; CDR H2, comprising the sequence shown in SEQ ID NO: 397; and CDR H3, comprising the sequence shown in SEQ ID NO: 399; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 411; CDR L2, which contains the sequence shown in SEQ ID NO: 413; and CDR L3, which contains the sequence shown in SEQ ID NO: 415. n) Heavy chain variable region (VH), comprising: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 427; CDR H2, comprising the sequence shown in SEQ ID NO: 429; and CDR H3, comprising the sequence shown in SEQ ID NO: 431; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 443; CDR L2, which contains the sequence shown in SEQ ID NO: 445; and CDR L3, which contains the sequence shown in SEQ ID NO: 447. o) Heavy chain variable region (VH), comprising: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 459; CDR H2, comprising the sequence shown in SEQ ID NO: 461; and CDR H3, comprising the sequence shown in SEQ ID NO: 463; and The light chain variable region (VL) comprises: CDR L1, containing the sequence shown in SEQ ID NO: 475; CDR L2, containing the sequence shown in SEQ ID NO: 477; and CDR L3, containing the sequence shown in SEQ ID NO: 479; or p) Heavy chain variable region (VH), comprising: complementarity-determining region (CDR) H1, comprising the sequence shown in SEQ ID NO: 491; CDR H2, comprising the sequence shown in SEQ ID NO: 493; and CDR H3, comprising the sequence shown in SEQ ID NO: 495; and The light chain variable region (VL) comprises: CDR L1, which contains the sequence shown in SEQ ID NO: 507; CDR L2, which contains the sequence shown in SEQ ID NO: 509; and CDR L3, which contains the sequence shown in SEQ ID NO: 511.

[0054] In another aspect, this disclosure provides a composition comprising one or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein the one or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 9; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 25; b) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 41; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 57; c) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 73; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 89; d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 105; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 121; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 137; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 153; f) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 185. g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 201; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 217. h) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 233; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 249. i) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 281. j) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 297; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 313; k) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 345; l) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 361; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 377. m) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 393; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 409; n) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 441. o) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 457; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 473; or p) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3, as shown in SEQ ID NO: 489; and the light chain variable region (VL) containing CDRs L1, L2 and L3, as shown in SEQ ID NO: 505.

[0055] In another aspect, this disclosure provides a composition comprising one or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein said one or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 75; a CDR H2, comprising the sequence shown in SEQ ID NO: 77; and a CDR H3, comprising the sequence shown in SEQ ID NO: 79; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 91; a CDR L2, comprising the sequence shown in SEQ ID NO: 93; and a CDR L3, comprising the sequence shown in SEQ ID NO: 95; b) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 171; CDR H2, comprising the sequence shown in SEQ ID NO: 173; and CDR H3, comprising the sequence shown in SEQ ID NO: 175; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 187; CDR L2, comprising the sequence shown in SEQ ID NO: 189; and CDR L3, comprising the sequence shown in SEQ ID NO: 191; c) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 267; a CDR H2, comprising the sequence shown in SEQ ID NO: 269; and a CDR H3, comprising the sequence shown in SEQ ID NO: 271; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 283; a CDR L2, comprising the sequence shown in SEQ ID NO: 285; and a CDR L3, comprising the sequence shown in SEQ ID NO: 287; d) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 299; CDR H2, comprising the sequence shown in SEQ ID NO: 301; and CDR H3, comprising the sequence shown in SEQ ID NO: 303; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 315; CDR L2, comprising the sequence shown in SEQ ID NO: 317; and CDR L3, comprising the sequence shown in SEQ ID NO: 319; e) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 331; CDR H2, comprising the sequence shown in SEQ ID NO: 333; and CDR H3, comprising the sequence shown in SEQ ID NO: 335; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 347; CDR L2, comprising the sequence shown in SEQ ID NO: 349; and CDR L3, comprising the sequence shown in SEQ ID NO: 351; f) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 395; a CDR H2, comprising the sequence shown in SEQ ID NO: 397; and a CDR H3, comprising the sequence shown in SEQ ID NO: 399; and the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 411; a CDR L2, comprising the sequence shown in SEQ ID NO: 413; and a CDR L3, comprising the sequence shown in SEQ ID NO: 415; or g) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 427; CDR H2, comprising the sequence shown in SEQ ID NO: 429; and CDR H3, comprising the sequence shown in SEQ ID NO: 431; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 443; CDR L2, comprising the sequence shown in SEQ ID NO: 445; and CDR L3, comprising the sequence shown in SEQ ID NO: 447.

[0056] In one embodiment of this disclosure, the composition further comprises two or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD). For example, the two or more antibodies or antigen-binding fragments may be selected from antibodies or antigen-binding fragments comprising: a) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 75; a CDR H2, comprising the sequence shown in SEQ ID NO: 77; and a CDR H3, comprising the sequence shown in SEQ ID NO: 79; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 91; a CDR L2, comprising the sequence shown in SEQ ID NO: 93; and a CDR L3, comprising the sequence shown in SEQ ID NO: 95; b) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 171; CDR H2, comprising the sequence shown in SEQ ID NO: 173; and CDR H3, comprising the sequence shown in SEQ ID NO: 175; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 187; CDR L2, comprising the sequence shown in SEQ ID NO: 189; and CDR L3, comprising the sequence shown in SEQ ID NO: 191; c) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 267; a CDR H2, comprising the sequence shown in SEQ ID NO: 269; and a CDR H3, comprising the sequence shown in SEQ ID NO: 271; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 283; a CDR L2, comprising the sequence shown in SEQ ID NO: 285; and a CDR L3, comprising the sequence shown in SEQ ID NO: 287; d) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 299; CDR H2, comprising the sequence shown in SEQ ID NO: 301; and CDR H3, comprising the sequence shown in SEQ ID NO: 303; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 315; CDR L2, comprising the sequence shown in SEQ ID NO: 317; and CDR L3, comprising the sequence shown in SEQ ID NO: 319; e) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 331; CDR H2, comprising the sequence shown in SEQ ID NO: 333; and CDR H3, comprising the sequence shown in SEQ ID NO: 335; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 347; CDR L2, comprising the sequence shown in SEQ ID NO: 349; and CDR L3, comprising the sequence shown in SEQ ID NO: 351; f) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 395; a CDR H2, comprising the sequence shown in SEQ ID NO: 397; and a CDR H3, comprising the sequence shown in SEQ ID NO: 399; and the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 411; a CDR L2, comprising the sequence shown in SEQ ID NO: 413; and a CDR L3, comprising the sequence shown in SEQ ID NO: 415; or g) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 427; CDR H2, comprising the sequence shown in SEQ ID NO: 429; and CDR H3, comprising the sequence shown in SEQ ID NO: 431; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 443; CDR L2, comprising the sequence shown in SEQ ID NO: 445; and CDR L3, comprising the sequence shown in SEQ ID NO: 447.

[0057] In one embodiment of this disclosure, the composition further comprises three or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD). For example, the one or more antibodies or antigen-binding fragments may be selected from antibodies or antigen-binding fragments thereof comprising: a) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 75; a CDR H2, comprising the sequence shown in SEQ ID NO: 77; and a CDR H3, comprising the sequence shown in SEQ ID NO: 79; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 91; a CDR L2, comprising the sequence shown in SEQ ID NO: 93; and a CDR L3, comprising the sequence shown in SEQ ID NO: 95; b) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 171; CDR H2, comprising the sequence shown in SEQ ID NO: 173; and CDR H3, comprising the sequence shown in SEQ ID NO: 175; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 187; CDR L2, comprising the sequence shown in SEQ ID NO: 189; and CDR L3, comprising the sequence shown in SEQ ID NO: 191; c) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 267; a CDR H2, comprising the sequence shown in SEQ ID NO: 269; and a CDR H3, comprising the sequence shown in SEQ ID NO: 271; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 283; a CDR L2, comprising the sequence shown in SEQ ID NO: 285; and a CDR L3, comprising the sequence shown in SEQ ID NO: 287; d) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 299; CDR H2, comprising the sequence shown in SEQ ID NO: 301; and CDR H3, comprising the sequence shown in SEQ ID NO: 303; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 315; CDR L2, comprising the sequence shown in SEQ ID NO: 317; and CDR L3, comprising the sequence shown in SEQ ID NO: 319; e) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 331; CDR H2, comprising the sequence shown in SEQ ID NO: 333; and CDR H3, comprising the sequence shown in SEQ ID NO: 335; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 347; CDR L2, comprising the sequence shown in SEQ ID NO: 349; and CDR L3, comprising the sequence shown in SEQ ID NO: 351; f) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 395; a CDR H2, comprising the sequence shown in SEQ ID NO: 397; and a CDR H3, comprising the sequence shown in SEQ ID NO: 399; and the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 411; a CDR L2, comprising the sequence shown in SEQ ID NO: 413; and a CDR L3, comprising the sequence shown in SEQ ID NO: 415; or g) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 427; CDR H2, comprising the sequence shown in SEQ ID NO: 429; and CDR H3, comprising the sequence shown in SEQ ID NO: 431; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 443; CDR L2, comprising the sequence shown in SEQ ID NO: 445; and CDR L3, comprising the sequence shown in SEQ ID NO: 447.

[0058] On the other hand, this disclosure provides a composition comprising one or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein said one or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 73; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 89; b) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 185. c) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 281. d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 297; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 313; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 345. f) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 393; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 409; or g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 441.

[0059] On the other hand, this disclosure provides a composition comprising a combination of two or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein the two or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 73; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 89; b) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 185. c) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 281. d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 297; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 313; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 345. f) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 393; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 409; or g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 441.

[0060] On the other hand, this disclosure provides a composition comprising a combination of three or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein the three or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 73; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 89; b) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 185. c) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 281. d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 297; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 313; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 345. f) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 393; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 409; or g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 441.

[0061] In one embodiment of this disclosure, the composition comprises a combination of three or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD). For example, the three or more antibodies or antigen-binding fragments may be selected from antibodies or antigen-binding fragments thereof comprising: a) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 73; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 89; b) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 137; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 153. c) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 185. d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 233; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 249; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 281. f) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 345. g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 393; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 409. h) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 444; or i) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 489; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 505.

[0062] In some embodiments of this disclosure, the composition comprises a combination of three or more recombinant antibody or antigen-binding fragments, wherein the selected three or more recombinant antibody or antigen-binding fragments maximize the ADP activity, or agglutination activity, or ADP activity and agglutination activity of the combination compared to ADP or agglutination of a single antibody or antigen-binding fragment.

[0063] In some embodiments of this disclosure, any of the foregoing compositions further comprises a pharmaceutically acceptable carrier.

[0064] In another aspect, this disclosure provides nucleic acid molecules encoding antibodies or antibody- or antigen-binding fragments as described in the foregoing aspects. In another aspect, this disclosure provides vectors comprising the nucleic acid molecules described in this disclosure. In some embodiments of this disclosure, the vector is an expression vector. In another aspect, this disclosure provides a host cell comprising the vector described in this disclosure. In some embodiments of this disclosure, the host cell is a prokaryotic or eukaryotic cell. In some embodiments of this disclosure, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, and other cells suitable for preparing antibodies or their antigen-binding fragments. In some embodiments of this disclosure, the mammalian cell is a CHO cell, a HEK293 cell, or a COS cell.

[0065] In another aspect, this disclosure provides a method for producing an antibody or an antigen-binding fragment thereof, wherein the host cells of this disclosure are cultured under conditions suitable for expressing nucleic acids encoding the antibody or antigen-binding fragment thereof, optionally wherein the antibody or antigen-binding fragment thereof is isolated, and optionally wherein the generated antibody or antigen-binding fragment thereof is collected.

[0066] In another aspect, this disclosure provides a method for treating or preventing allogeneic immunization of Rh-negative subjects in rhesus monkeys, wherein the method comprises administering to the desired Rh-negative subject an effective amount of the anti-Rh(D) antibody or antigen-binding fragment of this disclosure, or a combination thereof, or a composition described in this disclosure.

[0067] In some embodiments of this disclosure, the subject is pregnant.

[0068] In some embodiments of this disclosure, the method prevents hemolytic diseases in fetuses and newborns.

[0069] On the other hand, this disclosure provides the use of the antibodies or antigen-binding fragments or combinations thereof disclosed herein in the preparation of medicaments for the treatment or prevention of hemolytic diseases in fetuses and newborns.

[0070] On the other hand, this disclosure provides the use of the antibodies or antigen-binding fragments of this disclosure, or combinations thereof, or compositions thereof, for the treatment or prevention of allogeneic immunization in Rh-negative subjects in rhesus monkeys.

[0071] On the other hand, this disclosure provides a method for analyzing the most abundant antibodies binding to an antigen (RhD) in a polyclonal (pAb) population, wherein the method comprises: (a) performing proteomic analysis on a mixture of RhD-specific IgG from plasma, wherein the plasma is provided by a donor; affinity purifying a total IgG mixture from said plasma; and (b) reacting with RhD... +RBC or RhD - RBC incubation is used to isolate RhD-specific IgG subsets and non-RhD-specific IgG subsets, respectively; the RhD-specific IgG subsets and non-RhD-specific IgG subsets are independently fragmented into short peptides with overlapping sequences using one or more proteases; the fragmented peptides are analyzed by mass spectrometry to identify the sequence of each peptide; the sequences of the short peptides are assembled into longer peptide sequences belonging to a single antibody, optionally using computational software; antibodies derived from non-RhD-specific subsets are excluded and not further analyzed; and (b) BCR sequencing, wherein the provided B cells are isolated from the donor's blood; RNA is extracted from the B cells; the RNA is sequenced by next-generation sequencing (NGS) to provide transcriptomic sequence information; and the transcriptomic sequence information is combined with the proteomic sequence information to further assemble and obtain the complete amino acid sequence of the most abundant mAb in the RhD-specific IgG subset. In some preferred embodiments, the method for analysis further includes sorting the sequences by quantified enrichment fold changes of the RhD-specific IgG subsets to identify the most abundant mAbs that are specifically enriched in both the LC and HC regions.

[0072] In another aspect of this disclosure, a method is provided for analyzing the most abundant antibodies binding to an antigen (RhD) in a polyclonal (pAb) population, the method comprising: a) performing proteomic analysis on a mixture of RhD-specific IgG in plasma to provide proteomic sequence information; b) BCR sequencing; c) combining transcriptomic sequence information with the proteomic sequence information to further assemble and obtain the complete amino acid sequence of the most abundant mAb in the RhD-specific IgG subset; d) further selecting mAb combinations from the most abundant mAbs, wherein each mAb contains a different gene and / or amino acid sequence, and each mAb contains a variable region (VH) of the IgG1 subclass heavy chain.

[0073] In some embodiments of this disclosure, blood is collected from an RhD antigen donor. In some embodiments of this disclosure, donor plasma is isolated from PBMCs. In further embodiments of this disclosure, CD19... + IgG + B cells were isolated from donor PBMCs.

[0074] In some embodiments of this disclosure, the identified one or more amino acid sequences can be used to prepare recombinant antibodies or antigen-binding fragments that can be used in combination with one or more antibody or antigen-binding fragments disclosed herein. Attached Figure Description

[0075] Figure 1This is a schematic diagram illustrating the discovery of anti-RhD antibodies using a combination of next-generation sequencing (NGS) and proteomics.

[0076] Figure 2 This is a schematic diagram illustrating the cloning of a variable gene for an antibody and its cloning into an expression vector. (Regarding RhD...) + RBC and RhD - The specificity of purified antibodies was assessed in the agglutination assays of both RBCs.

[0077] Figure 3 This is a schematic diagram of the RhD antigen in the red blood cell membrane.

[0078] Figure 4 The agglutination results (RBCs treated with bromelain or untreated RBCs) are shown.

[0079] Figure 5 shows the competitive binding profiles of anti-RhD mAbs. A) Competitive RBC binding assay. The potency of each mAb is assessed based on its ability to inhibit the RhD binding of other anti-RhD mAbs. The number of inhibited mAbs refers to the number of antibodies whose RhD binding is inhibited by at least 70% (B) or 50% (C) when using the unconjugated mAb being tested.

[0080] Figure 6 illustrates the binding of anti-RhD mAb to different epitope RhD variants (A). Potential epitopes of anti-RhD mAb are shown based on the availability of epitopes in the tested RhD variants (B).

[0081] Figure 7 illustrates the inhibitory activity of RhD-pIgG-1 against RhD mAb binding, as determined by FACS analysis. A) The binding activity of each individual mAb against RhD in the absence or presence of other IgG products. + RBC binding. B) The bar chart shows the percentage of binding inhibition mediated by other IgG products, based on the reduced AUC of mAb binding in the absence of alternative IgG products.

[0082] Figure 8 illustrates the RBC clearance induced by anti-RhD mAb via THP-1 monocytes. A) Gating strategy used to analyze in vitro phagocytosis assays in the presence of non-RhD mAb (top panel) or anti-RhD mAb (bottom panel). B) Fcγ receptors on THP-1 monocytes; C) Phagocytosis by anti-RhD mAb; F) Bar graph depicting the area under the curve (AUC) of phagocytosis scores at different mAb concentrations; E) Subclass switching of clone 21a (from IgG2 to IgG1) and clone 104 (from IgG3 to IgG1).

[0083] Figure 9 illustrates RBC clearance induced by anti-RhD mAb via ADCC. A) Schematic diagram of the ADCC assay. B) ADCC of anti-RhD mAb. C) Subclass conversion of clone 21a (from IgG2 to IgG1) and clone 104 (from IgG3 to IgG1).

[0084] Figure 10 illustrates the activation of NK cells by anti-RhD mAb. A) Cytokines secreted by NK cells in the presence of anti-RhD antibody were detected using a Bioplex kit. B) Markers of NK cell activation were assessed using FACS in the presence of anti-RhD.

[0085] Figure 11 Germline usage of RhD heavy chains was demonstrated.

[0086] Figure 12 The use of germline cells with anti-RhD light chains was demonstrated.

[0087] Figure 13 This is an alphafold diagram modeling the interaction between anti-RhD mAb and RhD antigen. The RhD antigen is depicted as a coiled structure (the bottom molecule in each diagram). The antibody heavy and light chains are represented as ribbon-like structures (the top molecule in each diagram).

[0088] Figure 14 shows the predicted interactions between epitopes and RhD antigens. A) A model where RhD antigens are shown in dark gray and anti-RhD mAb epitopes are highlighted in light gray; B) Predicted anti-RhD mAb epitopes on RhD antigens are shown in circles.

[0089] Figure 15 The target sites of anti-RhD mAbs on the extracellular RhD loop are shown. The alphafold predicted binding sites for each mAb are represented by rectangles. The four mAbs interact with ASP350 (represented by black ellipses).

[0090] Figure 16 The modified anti-RhD mAb RBC binding was demonstrated.

[0091] Figure 17 This study demonstrates that FC-modified anti-RhD mAb activates NK cells and clears RBCs via ADCC. A) Gating strategies used to assess NK cell activation. RhD + RBCs were incubated with mAb. B) The bar chart shows CD107. + Cells, IFNγ + Or the percentage of total activated NK cells. C) RhD cells opsonized with anti-RhD antibodies. +ADCC test results using PBMCs depleted of monocytes in the presence of RBCs.

[0092] Figure 18 The ADP activity results of the Fc-modified anti-RhD mAb are shown. A) ADP activity (ADP score) of antibody variants at different concentrations. B) Area under the curve (AUC).

[0093] Figure 19 Demonstrates RhD mediated by anti-RhD antibody variants + RBC clearance. Antibody (1 mg / mL) against bromelain-treated (A) or untreated (B) RhD. + The ADCC activity of RBCs is shown in the figure above. Antibody (100 ng / mL) against RhD treated with bromelain (C) or untreated (D) was compared. + The ADP activity of RBCs is also shown in the figure below.

[0094] Figure 20 Demonstrates RhD mediated by anti-RhD antibody pool + RBC clearance. Antibody (1 mg / mL) against bromelain-treated (A) or untreated (B) RhD. + The ADCC activity of RBCs is shown in the figure above. Antibody pools (100 ng / mL) against bromelain-treated (C) or untreated (D) RhD... + The ADP activity of RBCs is shown in the figure below.

[0095] Figure 21 This demonstrates RBC binding of the anti-RhD antibody fragment.

[0096] Sequence List Index Table 1: Antibody sequences of clones identified by the method of the present invention

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] Detailed Implementation

[0118] Before describing the invention in detail, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0119] References to patent documents or other prior art herein should not be construed as an admission that such document or material was known prior to the priority date of any claim, or that the information contained therein was part of common general knowledge prior to the priority date of any claim.

[0120] Conventional techniques and definitions Throughout this specification, unless otherwise specifically stated or required by the context, references to a single step, substance composition, group of steps, or group of substance compositions shall be considered to include one or more (i.e., one or more) of such steps, substance compositions, group of steps, or group of substance compositions.

[0121] Those skilled in the art will understand that this disclosure is open to change and modification, except for the specific descriptions. It should be understood that this disclosure includes all such changes and modifications. This disclosure also includes all steps, features, compositions, and compounds individually or collectively mentioned or specified in this specification, as well as any two or more, or any and all combinations of said steps or features.

[0122] The scope of this disclosure is not limited to the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of this disclosure.

[0123] Unless otherwise specifically stated, any embodiment of this disclosure should be considered herein as applicable to any other embodiment of this disclosure with necessary modifications. In other words, any specific embodiment of this disclosure can be combined with any other specific embodiment of this disclosure (except where mutually exclusive).

[0124] Any embodiment of this disclosure that discloses a specific feature or feature group or method or method step should be regarded as providing explicit support for abandoning a specific feature or feature group or method or method step.

[0125] Unless otherwise specifically defined, all technical and scientific terms used herein should be assumed to have the same meaning as commonly understood by one of ordinary skill in the art. In this invention, standard procedures are well known to those skilled in the art. Such techniques have been described and explained in various publications, such as: J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); TA Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); DM Glover and BD Hames (ed.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); FM Ausubel et al. (ed.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date); Ed Harlow and David Lane (ed.), Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory (1988); and JE Coligan. et al., (edited) Current Protocols in Immunology, JohnWiley & Sons (including all updates to date).

[0126] The descriptions and definitions of variable regions and their portions, antibodies and their fragments in this article can be further clarified through discussion in the following: Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987 and 1991), Bork et al., J Mol. Biol. (1994) 242:309-320, 1994; Chothia and Lesk, J. Mol. Biol. (1987) 196:901-917; Chothia et al. Nature (1989) 342:877-883 and Al-Lazikani et al., J. Mol. Biol. (1997) 273:927-948.

[0127] The term “and / or”, such as “X and / or Y”, should be understood to mean “X and Y” or “X or Y”, and should be regarded as providing explicit support for both meanings or either meaning.

[0128] As used herein, unless the context explicitly states otherwise, the singular indefinite articles (“a”, “an”) and definite articles (“the”) include plural indicators. Thus, for example, references to antibody or antigen-binding fragments include combinations of two or more such molecules.

[0129] Throughout this specification, the word “comprise” or variations thereof, such as “comprises” or “comprising”, shall be understood to imply inclusion of the stated element, integer or step, or group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

[0130] As used in this article, the term “derived from” should be considered as indicating that the specified element (integer) can be obtained from a particular source, although it may not necessarily be directly derived from that source.

[0131] As used herein, the term "specific binding" should be considered as indicating that the interaction between binding regions on an antibody or its binding fragment depends on the presence of an antigenic determinant or epitope. Even when present in a mixture of other molecules or organisms, the binding region preferentially binds to or recognizes a specific antigenic determinant or epitope. In one instance, the reaction or association of a binding region with a particular component or a cell expressing it is more frequent, rapid, longer-lasting, and / or has greater affinity than its reaction or association with a substitute antigen or cell. By reading this definition, it should also be understood that, for example, a binding region that specifically binds to a particular component may specifically bind to or may not specifically bind to a second antigen. Therefore, "specific binding" does not necessarily preclude binding to another antigen or undetectable binding. In this document, the term "specific binding" may be used interchangeably with "selective binding." Generally, references to binding herein indicate specific binding, and each term should be understood to provide explicit support for the other. Methods for determining specific binding will be apparent to those skilled in the art. For example, a binding protein containing a binding region of this disclosure comes into contact with said component or a cell expressing said component or a mutant form of it or a substitute antigen. Then, the binding to the component, mutant form, or alternative antigen is determined, and the binding region as described above is considered to specifically bind to the component.

[0132] The term "recombinant" should be understood to refer to the product of artificial genetic recombination. Therefore, in the context of antibodies or their antigen-binding fragments, this term does not include antibodies naturally present in the subject's body as products of natural recombination occurring during B cell maturation. However, if such antibodies are isolated, they are considered isolated proteins containing the antibody's variable region. Similarly, if the nucleic acid encoding the protein is isolated and expressed using a recombinant approach, the resulting protein is a recombinant protein. Recombinant proteins also include proteins expressed through artificial recombination in cells, tissues, or subjects (e.g., the cells, tissues, or individuals in which they are expressed).

[0133] The term “polypeptide” or “polypeptide chain” should be understood as referring to a series of consecutive amino acids linked by peptide bonds.

[0134] Those skilled in the art will understand that an "antibody" is generally considered to be a protein comprising a variable region consisting of multiple polypeptide chains (e.g., a polypeptide containing a light chain variable region (VL) and a heavy chain variable region (VH)). Antibodies typically also contain constant domains, some of which can be arranged into a constant region; in the case of a heavy chain, the constant region comprises a constant fragment or crystallizable fragment (Fc). VH and VL interact to form an Fv containing an antigen-binding region capable of specifically binding one or more closely related antigens. Typically, light chains from mammals are κ or λ light chains, and heavy chains from mammals are α, δ, ε, γ, or μ. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In the context of this invention, the term "antibody" includes human antibodies. The term "antibody" also includes variants, such as variants lacking the encoding a C-terminal lysine residue, deamidated variants, and / or glycosylated variants, and / or variants containing a pyroglutamate salt (e.g., at the N-terminus), and / or variants lacking an N-terminal residue (e.g., in the antibody or V region and / or variants containing all or part of the secretion signal). Deamidated variants encoding asparagine residues can result in the production of isoaspartic acid and aspartic acid isoforms, or even succinamide involving adjacent amino acid residues. Deamidated variants encoding glutamine residues can produce glutamate. When a particular amino acid sequence is referred to, it is intended to include compositions comprising a heterogeneous mixture of such sequences and variants.

[0135] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in its essentially intact form, as opposed to the antigen-binding fragment of an antibody. Specifically, whole antibodies include those that have both a heavy chain and a light chain, including an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or a variant of its amino acid sequence.

[0136] As used herein, the term "monoclonal antibody (mAb)" refers to an antibody obtained from a substantially homogeneous population of antibodies or to a population of said antibodies. Individual antibodies comprising said populations are substantially identical, except that they may be present in small amounts as naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. This disclosure provides recombinant DNA expression of monoclonal antibodies. Two or more recombinant monoclonal antibodies (e.g., antibodies targeting different antigenic sites) can be mixed and used in the methods of this disclosure. Specific combinations or pools can be selected to provide a variety of recombinant antibodies, wherein each recombinant antibody can bind to one or more different rhesus monkey D antigenic epitopes.

[0137] As used herein, “human antibody” includes antibodies that have the amino acid sequence of human immunoglobulins and antibodies isolated from human immunoglobulin libraries.

[0138] As used herein, a "chimeric antibody" refers to a structural chimera having, for example, a variable sequence derived from one species and a constant region from another species or a constant region from another isotype within the same species. As used herein, a "variable region" refers to a portion of the light and / or heavy chain of an antibody as defined herein, capable of specifically binding to an antigen, and including, for example, the amino acid sequence of a complementarity-determining region (CDR) (i.e., CDR1, CDR2, and CDR3) and a framework region (FR). For example, a variable region includes three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0139] As used herein, the term “complementarity-determining region” (synonymous with CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the variable region of an antibody that result in specific antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. In one instance, the amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991. For example, according to Kabat’s numbering system, the FRs and CDRs of VH are located as follows: residues 1-30 (FR1), residues 31-35 (CDR1), residues 36-49 (FR2), residues 50-65 (CDR2), residues 66-94 (FR3), residues 95-102 (CDR3), and residues 103-113 (FR4). According to the Kabat numbering system, the FRs and CDRs of VL are located as follows: residues 1-23 (FR1), residues 24-34 (CDR1), residues 35-49 (FR2), residues 50-56 (CDR2), residues 57-88 (FR3), residues 89-97 (CDR3), and residues 98-107 (FR4). This disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above. In one embodiment, references to CDR (or FR) herein refer to those regions according to the Kabat numbering system.

[0140] The "frame region" (FR) consists of variable region residues other than CDR residues.

[0141] As used herein, the term "Fv" should be considered to refer to any protein in which VL and VH associate and form a complex having an antigen-binding site (i.e., capable of specifically binding to an antigen), whether composed of multiple polypeptides or a single polypeptide. The VH and VL forming the antigen-binding site may be in a single polypeptide chain or in different polypeptide chains. Furthermore, the Fv of this disclosure (and any protein of this disclosure) may have multiple antigen-binding sites, which may bind to the same antigen or may not bind to the same antigen. The term should be understood to include fragments directly derived from antibodies and corresponding proteins of such fragments generated using a recombinant approach. In some embodiments, VH is not linked to a heavy chain constant domain (CH), such as CH1, and / or VL is not linked to a light chain constant domain (CL). Exemplary peptides or proteins containing Fv include Fab fragments, Fab' fragments, F(ab') fragments, scFv, diabody, triabody, tetrabody, or higher-order complexes, or any of the foregoing linked to a constant region or its domain (e.g., one or both of the CH2 or CH3 domains), such as minibody.

[0142] "Fab fragments" consist of monovalent antigen-binding fragments of immunoglobulins and can be produced by digesting whole antibodies with papain to generate fragments consisting of a portion of the complete light chain and heavy chain, or by using recombinant methods.

[0143] The "Fab' fragment" of an antibody can be obtained by treating the whole antibody with pepsin and then reducing it to produce a molecule consisting of a complete light chain and a portion of a heavy chain containing VH and a single CH. Treating each antibody in this way yields two Fab' fragments. Fab' fragments can also be generated via recombinant synthesis.

[0144] The “F(ab')2 fragment” consists of a dimer of two Fab' fragments linked together by two disulfide bonds and is obtained by treating a whole antibody molecule with pepsin without subsequent reduction. The “Fab2” fragment is a recombinant fragment containing two Fab fragments linked by, for example, a leucine zipper or a CH3 domain.

[0145] "Single-chain Fv" or "scFv" is a recombinant molecule containing an antibody variable region fragment (Fv), in which the light chain variable region and the heavy chain variable region are covalently linked by a suitable flexible peptide linker.

[0146] As used herein, the term "constant region" refers to the portion of the antibody's heavy or light chain excluding the variable region. In the heavy chain, the constant region typically contains multiple constant domains and hinge regions; for example, the IgG constant region contains components linked by CH1, hinge, CH2, and CH3. In the heavy chain, the constant region contains the Fc region. In the light chain, the constant region typically contains a single constant domain (CL1).

[0147] The terms “crystallizable fragment” or “Fc” or “Fc region” or “Fc moiety” (these terms are used interchangeably herein) refer to a region of an antibody that contains at least one constant domain, and that region is typically (but not necessarily) glycosylated and capable of binding one or more Fc receptors and / or components of the complement cascade. The heavy chain constant region can be selected from any of five isotypes: α, δ, ε, γ, or μ. Furthermore, multiple subclasses of the heavy chain (e.g., subclasses of the IgG heavy chain) are responsible for different effector functions; therefore, by selecting the desired heavy chain constant region, a protein with the desired effector function can be produced. Exemplary heavy chain constant regions are γ1 (IgG1), γ2 (IgG2), and γ3 (IgG3), or hybrids thereof.

[0148] The terms “antigen-binding portion of an antibody,” “antigen-binding fragment,” “antigen-binding domain,” “antibody fragment,” or “functional fragment of an antibody” are used interchangeably in this disclosure to refer to one or more antibody fragments that retain the ability to specifically bind antigens (see generally Holliger et al., Nature Biotech. (2005) 23 (9):1126-1129). Non-limiting examples of antibody fragments include: (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds in a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody single arm; (v) dAb fragments (Ward et al., Nature (1989) 341:544 546) consisting of a VH domain; and (vi) separated complementarity-determining regions (CDRs). Furthermore, although the two domains VL and VH of an Fv fragment are encoded by different genes, they can be linked together using recombinant methods via synthetic adapters to form a single protein chain, where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)) (see, for example, Bird et al., Science (1988) 242:423 426; and Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879 5883; and Osbourn et al., Nat. Biotechnol. (1998) 16:778). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a particular scFv can be linked with human immunoglobulin constant region cDNA or genomic sequences to produce expression vectors encoding complete IgG molecules or other isotypes. VH and VL can also be used to generate other fragments of Fab, Fv, or immunoglobulins using protein chemistry or recombinant DNA techniques. Other forms of single-chain antibodies, such as bispecific antibodies, are also included.

[0149] As used herein, the term "subject" refers to a human being. The terms "subject," "patient," and "individual" are used interchangeably in this document.

[0150] As used herein, the term "patient" includes a living person who is receiving or should receive medical care due to a disease or condition. This includes subjects who are examined for pathological signs but have not been diagnosed with a disease or have no observable symptoms.

[0151] In some instances, the term "subject" refers to an Rh-negative subject (e.g., an Rh-negative pregnant subject) who is at risk of rhesus alloimmunization or has symptoms of rhesus alloimmunization. In other instances, "subject" refers to a fetus or newborn at risk of hemolytic disease due to the mother's rhesus alloimmunization.

[0152] As used herein, a subject at “risk” of having a disease or condition or its recurrence or relapse may or may not have detectable symptoms, and may or may not show detectable symptoms prior to treatment according to this disclosure. “At risk” means that the subject has one or more risk factors that are measurable parameters related to the development of the disease or condition, as known in the art and / or described herein.

[0153] As used herein, the term "treatment" ("treating", "treat", or "treatment") includes the administration of an antibody or antigen-binding fragment of this disclosure to reduce or eliminate at least one symptom of a disease or condition or to slow the progression of a disease or condition. Anti-D antibodies may be used, for example, as prophylactic treatment and / or therapeutic treatment for potential sensitization events in Rh-negative women who are pregnant or recently pregnant (e.g., up to 10 days after the cessation of pregnancy).

[0154] As used herein, the term "preventing" ("prevent" or "prevention") includes providing prophylaxis for the onset or recurrence of a disease or condition. An individual may have a predisposition or risk of developing or experiencing a disease or recurrence, but has not yet been diagnosed with the disease or the recurrence.

[0155] "Effective amount" refers to an amount that effectively achieves the desired result at least within a certain dose and necessary duration. For example, the desired result can be a therapeutic or preventative result. An effective amount can be provided in one or more administrations. In some instances of this disclosure, the term "effective amount" means the amount necessary to effectively treat a disease or condition. In some instances of this disclosure, the term "effective amount" means the depletion or elimination of RhD. + Red blood cells instead of RhD negative (RhD) -The amount of red blood cells required. This may be referred to in the art as RBC clearance. While not wishing to be theoretically limited, RBC clearance can be mediated by antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP). For a general description of the application of these methods in RBC clearance protocols, see Cruz-Leal et al. Prevention of haemolytic disease of the foetus and newborn: what have we learned from animal models? Current Opinion in Hematology 24(6):p 536-543, 2017. The effective amount can vary depending on the disease or condition to be treated or the factors to be modified, as well as on weight, age, ethnic background, sex, health and / or physical condition, and other factors relevant to the subject to be treated. Generally, the effective amount will fall within a relatively broad range (e.g., a “dosage” range) that can be determined by a medical practitioner through routine trials and experiments. Therefore, this term should not be construed as limiting this disclosure to a specific amount. The effective dose can be administered as a single dose, or as a dose that is repeated once or multiple times during the treatment period.

[0156] Antibody and antigen binding fragment This invention relates to antibodies against the RhD antigen on human erythrocytes. The rhesus monkey blood group system is a major antigenic component of the human erythrocyte membrane; in this blood group, the RhD antigen has specific clinical importance in alloimmune responses. Receiving RhD... + Blood contains anti-RhD RhD - Individuals may suffer from massive red blood cell (RBc) destruction due to Rh(D) phenotype incompatibility; therefore, donor blood must routinely be classified as RhD. + or RhD - .

[0157] The RhD antigen is also responsible for hemolytic disease of the newborn (HDN). This condition occurs in RhD cells that were previously sensitive to the RhD antigen. - "Mother's" newborn RhD + In infants, this is caused by IgG anti-RhD antibodies crossing the placenta during pregnancy and destroying fetal red blood cells (RBCs). Because some fetal RBCs enter the maternal circulation and are recognized as foreign substances by the maternal immune system, RhD... - The mother's sensitivity to the RhD antigen usually occurs after the previous RhD infection. + During the birth of the "child".

[0158] To reduce the incidence of HDN, the standard practice is to perform RhD...+ The baby was immediately transferred to RhD after birth. - The mother was given polyclonal anti-RhD antibodies to rapidly remove any RhD that might have entered the maternal circulation. + RBC (Mollison, PL, BloodTransfusion is clinical Medicine, 7th ed., Blackwell Scientific, Oxford, 1983; Laros Jr., RK, Erythroblastosis Fe-talis. Blood Group Disorders in Pregnancy, 1986, Ch.7, p. 103).

[0159] The anti-RhD antibody and antigen-binding fragment disclosed herein are monoclonal antibodies that can be used alone or in combination. The antibodies can be genetically engineered to enhance specific functional activity. In several embodiments, the antibody and antigen-binding fragment comprises all or part of the antibody's constant region. In some embodiments, the constant region is an isotype selected from IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), and IgM.

[0160] In some embodiments, the antibody or antigen-binding fragment is derived from germline genes including IGHV3-33, IGHV3-30, IGHV3-30.3, IGHV3-30.5, IGHV3-33, IGHV1-2, IGHV2-26, IGHV3-21, IGHV3-30, IGHV3-53, IGHV4-34, IGHV4-39, and IGHV4-59. In some embodiments, the IGHD segment of the antibody or antigen-binding fragment is derived from germline genes including IGHD1-26, IGHD2-2, IGHD2-21, IGHD3-3, IGHD3-9, IGHD3-10, IGHD3-16, IGHD3-22, IGHD5-12, IGHD5-18, IGHD6-19, and IGHD6-6. In some embodiments, the JH segment of the antibody or antigen-binding fragment is derived from germline genes including IGHJ4, IGHJ6, IGHJ3, and IGHJ5. In some embodiments, the light chain segment of the antibody or antigen-binding fragment is derived from germline genes including IGLV1-47, IGLV2-14, IGLV2-23, IGLV7-43, IGLV1-51, IGKV1-39, and IGKV2-28.

[0161] In specific implementations, the anti-RhD antibody disclosed herein may comprise multiple isotypes of the same antibody, or a combination of antibodies with different isotypes.

[0162] The anti-RhD antibody and antigen-binding fragment disclosed herein may include, for example, one or more CDRs (e.g., CDR3), a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof.

[0163] The antibody or antigen-binding fragment may include a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region and / or light chain variable region comprises at least one CDR amino acid sequence (e.g., CDR3) or VH sequence or VL sequence as defined herein, or a sequence having an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the aforementioned sequence.

[0164] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., a gap may be introduced into the first amino acid or nucleic acid sequence to optimize alignment with the second amino acid or nucleic acid sequence). The corresponding amino acid or nucleotide positions are then compared. The two molecules are considered identical at that position when the position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared positions (i.e., identity % = number of overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are of the same length.

[0165] The determination of the percentage of identity between two sequences can also be accomplished using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm in Karlin and Altschul, Proc. Natl. Acad. Sci. USA (1990) 87:2264-2268, and the algorithm modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA (1993) 90:5873-5877. Such algorithms are incorporated into the NBLAST and XBLAST procedures of Altschul et al., J.Mol. Biol. (1990) 215:403. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set, for example, score=100, word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed using the XBLAST procedure parameter set, for example, score-50, word length=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain vacancy alignments for comparative purposes, Gapped BLAST, as described in Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402, can be used. Alternatively, PSI-BLAST can be used for iterative searching to detect distant relationships between molecules (ibid.). When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., the default parameters for XBLAST and NBLAST) can be used (see, for example, the NCBI website). Another preferred, non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, CABIOS (1988) 4:11-17. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, a PAM 120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4 can be used.

[0166] The antibody or antigen-binding fragments disclosed herein can bind to epitopes, or can bind to RhD sequence variants and partial D epitopes as described in (VoxSang 1996;70:123-131). In some embodiments, the antibody or antigen-binding fragment contacts residues on RhD, including D40, R114, N162, D164, R229, S230, E233, D350, A354, N356, Q362, and / or E369. In a further embodiment, the CDRH3 loop plays a key role in epitope recognition. In a further embodiment, the CDRH3 loop consists of 16 to 22 amino acid lengths. In a further embodiment, antibody or antigen-binding fragments that interact with more residues on RhD have a stronger ability to inhibit the binding of other anti-RhD antibody or antigen-binding fragments.

[0167] Antibodies or binding fragments can be antibodies or fragments whose sequences are modified to insert one or more amino acids into one or more hypervariable regions, as described, for example, in Jung and Pluckthun, Protein Engineering (1997) 10:9, 959-966; Yazaki et al., Protein Eng. Des Sel. (2004) 17(5):481-9 and US 2007 / 0280931.

[0168] In some embodiments, the antibody or antigen-binding fragment comprises a constant region or a portion thereof, such as the Fc. The constant region or a portion thereof may contain one or more amino acid substitutions. For example, the Fc may contain one or more amino acid substitutions that alter its binding to the Fc receptor and / or effector function.

[0169] In one embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a particular embodiment, the Fc receptor is an activated human Fcγ receptor, more specifically, human FcγRIIIa, FcγRI, or FcγRIIa, most specifically, human FcγRIIIa. In one embodiment, the effector function is one or more selected from the group consisting of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and the secretion of cell death mediators such as cytokines or reactive oxygen species (ROS). In one embodiment, the effector function is ADCC.

[0170] In some embodiments, the Fc cell is modified by glycosylation, particularly by fucosylation and / or galactosylation. The degree and type of glycosylation have been shown to affect antibody activity (Kumpel et al. HumAntibodies Hybridomas. 1994;5(3-4):143-51.; Sibéril et al Clin Immunol. 2006 Feb-Mar;118(2-3):170-9.). In some embodiments, the glycosylation pattern can be modified by chemical or enzymatic modification. In some embodiments, this modification can be achieved through Fc mutation, glycoengineering, and subclass switching.

[0171] In some implementations, the modification can be an antibody Fc mutant variant, which may include mutations in IgG1-GASDALIE such as G236A, S239D, A330L, and I332E (IgG1-GASDALIE antibodies), and G236R and L328 (IgG1-GRLR antibodies). Other modifications may be those mentioned in Edwards et al. Enhancement of Antibody-Dependent Cellular Cytotoxicity and Phagocytosis in Anti-HIV-1 Human-Bovine Chimeric Broadly Neutralizing Antibodies. J Virol., 95(13, 2021) or Bournazos et al. Broadly neutralizing anti-HIV-1 antibodies require Fc effector functions for in vivoactivity. Cell. 2014 Sep 11;158(6):1243-1253.

[0172] Antibody and antigen-binding fragment activity This invention relates to the ability to specifically bind to RhD-positive (RhD) antibodies. + RBC but cannot bind to RhD negative (RhD) - Antibody or antigen-binding fragments of RhD. Advantageously, the antibody or antigen-binding fragment can agglutinate RhD. + Red blood cells but cannot agglutinate RhD - Red blood cells. In some implementations, the antibody or antigen-binding fragment is associated with RhD. + The strength of RBC binding and its induction of RhD +The ability of RBCs to agglutinate is relevant. Antibody or antigen-binding fragments can be able to induce one or more of the following: RhD... + Phosphatidylserine exposure on RBCs and binding to annexin V; specifically in RhD + RBC induces the production of reactive oxygen species (ROS); increases RhD. + RBC size; change RhD + Protein expression levels in RBCs; and upregulation with RhD + Proteomics pathways related to cellular stress and lipolysis in RBCs.

[0173] Antibodies or antigen-binding fragments can also induce RhD. + RBC natural killer (NK) cell-mediated hemolysis. NK cell-mediated RhD induced by antibody or antigen-binding fragments. + RBC hemolysis may not be inhibited by caspase, cathepsins, RIPK1, RIPK2, or proteasome inhibitors. Furthermore, while not wishing to be limited by theory, antibodies or antigen-binding fragments can induce NK cell-mediated RhD by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). + RBC clearance, in which antibody or antigen-binding fragments bind to RhD + The strength of RBCs is correlated with their ability to induce ADCC. Antibodies or antigen-binding fragments can induce RhD independent of cell-to-cell contact. + RBC NK cell-mediated hemolysis. Antibodies or antigen-binding fragments can also induce ROS production in NK cells. Furthermore, RhD cells treated with antibodies or antigen-binding fragments... + RBC incubation can upregulate the production of cytokines and chemokines (e.g., one or more of RANTES, MIP-1a, MIP-1B, IL-8, IL-9, IL-17, IL-12, IL-1b, IFN-γ, TNF-α, CD69, NKP46, and NKG2D) in NK cells.

[0174] Antibody or antigen-binding fragments can also induce monocyte-mediated RhD. + RBC clearance. Induction of monocyte-mediated RhD. + RBC clearance can occur through the interaction between antibodies or antigen-binding fragments and Fcγ receptors.

[0175] Manufacturing method Methods for generating antibodies are known in the art and / or described in Harlow and Lane (ed.) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

[0176] Nucleic acids, recombinant vectors and host cells This disclosure includes nucleic acid molecules encoding variable regions of immunoglobulin light chains and / or heavy chains for anti-RhD antibodies, vectors containing such nucleic acids, and host cells capable of producing the anti-RhD antibodies of this disclosure. In some aspects, the nucleic acid molecules encode anti-RhD antibodies and antibody-binding fragments, as well as fusion proteins and chimeric antigen receptors containing them, and the host cells are capable of expressing anti-RhD antibodies and antibody-binding fragments, as well as fusion proteins and chimeric antigen receptors containing them.

[0177] The anti-RhD antibody of this disclosure can be prepared by recombinantly expressing genes encoding the light and heavy chains of immunoglobulins in host cells. To recombinantly express the antibody, host cells are transfected with one or more recombinant expression vectors carrying DNA fragments encoding the antibody immunoglobulin light and heavy chains, causing the light and heavy chains to be expressed in the host cells and optionally secreted into a culture medium from which the antibody can be recovered. Standard recombinant DNA methods are used to obtain the genes for the antibody heavy and light chains, to integrate these genes into recombinant expression vectors, and to introduce the vectors into host cells, such as those described in *Molecular Cloning; A Laboratory Manual, 2nd Edition* (Sambrook, Fritsch and Maniatis (eds.), Cold Spring Harbor, NY, 1989), *Current Protocols in Molecular Biology* (Ausubel, FM et al., eds., Greene Publishing Associates, 1989), and U.S. Patent No. 4,816,397.

[0178] To generate nucleic acids encoding such anti-RhD antibodies, DNA fragments encoding the variable regions of the light and heavy chains must first be obtained. These DNAs can be obtained by amplifying and modifying germline DNA or cDNA encoding the variable sequences of the light and heavy chains, for example, using polymerase chain reaction (PCR). Germline DNA sequences of the variable region genes of the human heavy and light chains are known in the art (see, for example, the “VBASE” Human Germline Sequence Database; see also Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. (1992) 22T:116-198 and Cox et al., Eur. J. Immunol. (1994) 24:827-836).

[0179] Once DNA fragments encoding the anti-RhD antibody-associated VH and VL regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VH or VL is operatively linked to another DNA fragment encoding a different protein (e.g., an antibody constant region or a flexible linker). As used in this context, the term "operatively linked" is intended to mean linking two DNA fragments such that the amino acid sequences encoded by both fragments remain within the frame.

[0180] By operatively linking DNA encoding VH to another DNA molecule encoding heavy chain constant regions (CH1, CH2, CH3, and optionally CH4), isolated DNA encoding VH regions can be converted into full-length heavy chain genes. Sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments containing these regions can be obtained by standard PCR amplification. Heavy chain constant regions can be constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but in some embodiments are constant regions of IgG1 or IgG4. For Fab fragment heavy chain genes, DNA encoding VH can be operatively linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0181] By operatively linking the DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated DNA encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ constant region or a λ constant region, but in some embodiments it is a κ constant region.

[0182] To create the scFv gene, the DNA fragments encoding VH and VL can be operatively linked to another fragment encoding a flexible linker (e.g., the amino acid sequence (Gly4~Ser)3), so that the VH and VL sequences can be expressed as a continuous single-stranded protein, with the VH and VL regions connected by the flexible linker (see, for example, Bird et al., Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0183] To express the anti-RhD antibody of this disclosure, DNA encoding the partial or full-length light and heavy chains, as described above, is inserted into an expression vector such that the gene is operatively linked to transcriptional and translational control sequences. In this context, the term "operatively linked" is intended to mean linking the antibody gene to a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the host cell used for expression. The antibody light chain gene and antibody heavy chain gene can be inserted into different vectors, or more typically, both genes can be inserted into the same expression vector.

[0184] The antibody gene is inserted into the expression vector using standard methods (e.g., linking the antibody gene fragment to a complementary restriction site on the vector, or blunt-end ligation if no restriction site is found). The expression vector may already carry the antibody constant region sequence before inserting the light or heavy chain sequence associated with the anti-RhD antibody. For example, one method of converting the VH and VL sequences associated with an anti-RhD monoclonal antibody into a full-length antibody gene is to insert them, respectively, into an expression vector that already encodes the heavy chain constant region and the light chain constant region, such that the VH segment is operatively linked to the CH segment within the vector, and the VL segment is operatively linked to the CL segment within the vector. Alternatively, the recombinant expression vector may encode a signal peptide that promotes the secretion of the antibody chain by host cells. The antibody chain gene can be cloned into the vector such that the signal peptide is linked within the frame to the N-terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a non-immunoglobulin protein).

[0185] In addition to the antibody chain gene, the recombinant expression vector of this disclosure also carries a regulatory sequence that controls the expression of the antibody chain gene in host cells. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. Those skilled in the art will understand that the design of the expression vector (including the selection of regulatory sequences) can depend on factors such as the selection of the host cell to be transformed, the desired protein expression level, etc. Suitable regulatory sequences for expression in mammalian host cells include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyomaviruses. For further description of viral regulatory elements and their sequences, see, for example, US 5,168,062, US 4,510,245 and US 4,968,615.

[0186] In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of this disclosure may also carry other sequences, such as sequences regulating vector replication in host cells (e.g., origin of replication) and selectable marker genes. Selectable marker genes facilitate selection in host cells to which the vector has been introduced (see, for example, US 4,399,216, US 4,634,665, and US 5,179,017). For example, typically, selectable marker genes confer resistance in host cells to drugs such as G418, hygromycin, or methotrexate. Suitable selectable marker genes include dihydrofolate reductase (DHFR) genes (for DHFR host cells with methotrexate selection / amplification) and neo genes (for G418 selection). To express the light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells using standard techniques. The term “transfection” is intended to encompass a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipid transfection, calcium phosphate precipitation, DEAE-dextran transfection, etc.

[0187] The antibodies disclosed herein can be expressed in prokaryotic or eukaryotic host cells. In some embodiments, antibody expression is performed in eukaryotic cells, such as mammalian host cells, that optimally secrete properly folded and immunologically active antibodies. Exemplary mammalian host cells for expressing the recombinant antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA (1980) 77:4216-4220, used with DHFR selective markers, for example, as described in Kaufman and Sharp, Mol. Biol. (1982) 159:601-621), Expi-CHO, NSO myeloma cells, COS cells, and SP2 cells. Other cell lines, such as the rat hybridoma cell line YB2 / 0, are also exemplary due to their ability to generate low-fucosylated antibodies (Teylaert et al, BMC Biotechnol 11(1) (2011)). In some implementations, the addition of supplements such as kifunensine or 2-F-thiacetized fucose, galactose, or dexamethasone to the culture medium can also affect glycosylation (Ehret et al, Biotechol Bioeng 116(4) (2019)). FUT8 KO CHO cells (Yang et al, Frontiers Chem, 9 (2021)) have also been shown to produce antibodies that are completely non-fucosylated. Other methods to reduce fucosylation include controlling culture conditions (Konno et al Cytotechnology, 64(3)(2012)).

[0188] The host cell can also be used to produce a portion of the complete antibody, such as a Fab fragment or an scFv molecule. It should be understood that changes to the above steps are within the scope of this disclosure. For example, it may be desirable to transfect the host cell with DNA encoding either the light chain or the heavy chain (but not both) of the anti-RhD antibody disclosed herein.

[0189] Once the anti-RhD antibody of this disclosure is generated through recombinant expression, it can be purified using any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., protein A affinity chromatography or protein G chromatography), size exclusion chromatography, ion exchange, centrifugation, differential solubility, or any other standard technique for protein purification. Following separation, the anti-RhD antibody can be further purified if desired, for example by high-performance liquid chromatography or by gel filtration chromatography.

[0190] When proteins are secreted into the culture medium, the supernatant from such expression systems can first be concentrated using a commercially available protein concentrate filter.

[0191] Protease inhibitors, such as PMSF, may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of foreign contaminants. Alternatively, or additionally, the supernatant may be filtered and / or separated from the protein-expressing cells, for example, using continuous centrifugation.

[0192] Furthermore, the anti-RhD antibody and / or binding fragment disclosed herein can be fused with the heterologous polypeptide sequence described herein or other sequences known in the art to facilitate purification. For example, multihistidine tags (such as hexahistidine tags), or influenza virus hemagglutinin (HA) tags, or simian virus 5 (V5) tags, or FLAG tags, or glutathione S-transferase (GST) tags. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexahistidine tag can be purified by contacting a sample containing the protein with nickel-nitrotriacetic acid (Ni-NTA) specifically bound to a hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and then eluting the bound protein. Alternatively, or further, a tag-bound ligand or antibody can be used in the affinity purification method.

[0193] Pharmaceutical Composition The anti-RhD antibodies and fragments disclosed herein may be in the form of compositions comprising one or more anti-RhD antibodies or binding fragments and one or more carriers, excipients, and / or diluents. The form of the composition (e.g., dry powder, liquid formulation, etc.) and the excipients, diluents, and / or carriers used will depend on the intended use of the antibody or fragment, and for therapeutic use, on the method of administration.

[0194] In a preferred embodiment, the composition comprises two or more anti-RhD antibodies or binding fragments. In a more preferred embodiment, the composition comprises three or more anti-RhD antibodies or binding fragments.

[0195] How to use The anti-RhD antibodies, binding fragments, or compositions disclosed herein can be used to treat or prevent neonatal hemolytic disease, idiopathic thrombocytopenic purpura (ITP), or RhD-negative individuals who have received RhD-positive blood transfusions.

[0196] In a preferred embodiment, the anti-RhD antibody, binding fragment, or composition of this disclosure can be used to prevent Rh-negative alloimmunization in Rh-negative individuals. Not wishing to be bound by theory, the mechanism of action involves the specific binding of the antibody or fragment thereof to the RhD-positive erythrocyte D antigen, followed by the removal of these erythrocytes from circulation, which is believed to occur primarily in the spleen. This clearance is associated with a dynamic mechanism that suppresses the individual's primary immune response, thereby preventing immunization.

[0197] Therefore, administration of the disclosed antibodies, conjugate fragments, or compositions to RhD-negative "mothers" is particularly useful for the prevention of neonatal hemolytic disease. The disclosed antibodies, conjugate fragments, or compositions can also be used prophylactically immediately after the birth of an Rh-positive child to prevent alloimmunization in RhD-negative women and to prevent neonatal hemolytic disease during subsequent pregnancies; and to prevent alloimmunization during miscarriage, ectopic pregnancy in RhD incompatibility cases, or transplacental hemorrhage caused by amniocentesis, chorionic villus sampling, and invasive obstetric procedures in rhesus D incompatibility cases.

[0198] Analysis of antibodies binding to the antigen (RhD) in donor plasma This invention utilizes a method for sequencing antibodies from donor blood that has been screened to express anti-RhD antibodies.

[0199] In some embodiments, donor blood is first separated into plasma and PBMC fractions by centrifugation. The total IgG contents can be purified by incubating the plasma fraction with, for example, protein G agarose. RhD-specific antibodies can be further isolated by incubating the total IgG pool with RhD-positive RBCs. Preferably, the antibody-bound RBCs are washed to remove non-specific conjugates, and then the antibodies are eluted with, for example, EDTA / glycine to produce an RhD-specific antibody pool. A similar selection step can be performed by incubating the total IgG pool with RhD-negative RBCs to isolate antibodies that bind to non-RhD antigens present on the RBCs. The RhD-specific antibody pool and the non-RhD-specific antibody pool can then be subjected to peptic digestion and mass spectrometry analysis to identify antibody sequences. Advantageously, sequences identified only in the RhD-specific pool (considered to be RhD-specific conjugates) can be used for further analysis.

[0200] The relative antibody transcription levels of B lymphocytes can be analyzed from PBMCs of donor blood. Active CD19 can be enriched from donor PBMCs using fluorescence-activated cell sorting (FACS). + IgG +Cells can then be sequenced for single-cell RNA. This type of sequencing offers the benefit of pairing the full-length heavy and light chain sequences of the expressed B-cell receptor (BCR). Combining the resulting transcriptomic data with proteomic analysis of RhD-specific binders allows for the identification of RhD-specific high-frequency, linked heavy and light chains.

[0201] Example This article describes a method for identifying and then preparing human monoclonal antibodies (mAbs) capable of activating effector cells (e.g., NK cells and THP-1 monocytes) and causing RBC clearance via ADCC and phagocytosis, respectively. The biological function of the resulting antibodies was then tested using a series of assays listed below and described in detail in the Experimental section.

[0202] i. Agglutination test (plate-based) ii. RBC binding assay (based on FACS) iii. Competitive binding test between mAb iv. Epitope mapping v. Counteracting the inhibition of RhD mAb binding with commercially available hyperimmune RhD-pIgG-1 vi. Phagocytosis assay (based on FACS) vii. ADCC (based on FACS) viii. NK cell activation assay (based on FACS) ix. Cytokine secretion assay Example 1: Generation and Characterization of Anti-RhD Antibodies Methods for identifying RhD-specific antibodies Blood collection and processing Peripheral blood was obtained from 27 “super producers” participating in the Australian Red Cross Lifeblood (ARCL) RhD project. Peripheral blood mononuclear cells (PBMCs) were purified from the blood using standard techniques (see, for example, Panda and Ravindran, 2013). Bio-protocol 3(3): and Panda et al 2012, PLoS Pathog 8(5)). RBCs were lysed from the final cell pellet, followed by washing of PBMCs with cold PBS and freezing at -80°C with heat-inactivated 90% fetal bovine serum and 10% dimethyl sulfoxide (DMSO).

[0203] B cell sorting (antibody nucleotide sequencing).

[0204] The samples were divided into 4 groups (5-11 samples per pool) and stained with BV605 anti-human IgG, APC-H7 CD19, and SYTOXGreen dead cell staining agent. Then, live CD19 samples were... + IgG + Cells were sorted and then every two samples were mixed again (finally n=2) for 10X Genomics gene sequencing (NGS).

[0205] Purify anti-RhD antibodies from donor plasma.

[0206] In this invention, donor blood is first separated into plasma and PBMC fractions by centrifugation. The total immunoglobulin fraction is purified by affinity chromatography (using protein G agarose). RhD-specific antibodies are further separated by incubating the total IgG pool with RhD-positive RBCs. The antibody-bound RBCs are washed to remove non-specific bindings, and then the antibodies are eluted with EDTA / glycine to produce a pool of RhD-specific antibodies.

[0207] Using proteomics to determine differential protein abundance The antibodies prepared above were analyzed using mass spectrometry-based proteomics. To obtain small peptides for further analysis, samples were prepared below by digestion with AspN, GluC, trypsin, and chymotrypsin.

[0208] For AspN, GluC, and trypsin digestion: Antibody (2 μg) was incubated with 50 mM ammonium bicarbonate, 8 M urea, and 1 mM DTT at 37°C for 30 min. The antibody was alkylated with 10 mM iodoacetamide for 1 h, followed by quenching with 10 mM DTT. The sample was diluted with 50 mM ammonium bicarbonate to reduce the UREA concentration to 1 M. 100 ng of trypsin, AspN, or GluC was added for digestion overnight at 37°C. The peptides were then desalted using a C18 STAGE tip and collected by centrifugation into new tubes. The collected peptides were lyophilized to dryness using a CentriVap (Labconco) and then reconstituted in 10 μL of 0.1% FA / 2% ACN for mass spectrometry analysis.

[0209] For chymotrypsin digestion: Antibody (2 μg) was incubated with 100 mM Tris-HCl pH 7.6, 8 M UREA, and 1 mM DTT at 37°C for 30 min. The antibody was alkylated with 10 mM iodoacetamide for 1 h, followed by quenching with 10 mM DTT. The sample was diluted with 100 mM Tris-HCl to reduce the UREA concentration to 1 M. 10 mM CaCl2 and 100 ng of chymotrypsin were added to each sample for digestion overnight at 37°C. The peptides were then desalted using a C18 STAGE tip and collected by centrifugation into new tubes. The collected peptides were lyophilized to dryness using a CentriVap (Labconco) and then reconstituted in 10 μL of 0.1% FA / 2% ACN for mass spectrometry analysis.

[0210] Mass spectrometry analysis Using nanoflow HPLC (M-class, Waters), the Ca filled into the emitter was analyzed. 18 Peptides (5 μL) were separated by reversed-phase chromatography on a molten silica column (IonOpticks).

[0211] Generate heavy chain and light chain IgG.fasta files Using 10x Genomics, a .fasta file was created from the output of CellRanger (all_contig_annotations.json) for subsequent MaxQuant retrieval. This .fasta file consists of barcodes, amino acid sequences, and nucleotide sequences, which were identified as high_confidence = TRUE and full_length = TRUE.

[0212] Raw data processing and analysis Raw data files were analyzed using the integrated Andromeda search engine in MaxQuant v1.6.17 software. The experiment type was set to TIMS-DDA, with no changes to the default settings. Strict trypsin, AspN, GluC, or chymotrypsin specificity was used, allowing a maximum of two missed cleavages. Data was retrieved based on an internally generated heavy / light chain IgG .fasta database and a separate reverse bait database. The minimum required peptide length was set to 7 amino acids. Modifications: Cys carbamoyl methylation was set as a fixed modification, while protein N-acetylation and Met oxidation were set as variable modifications. The initial peptide tolerance was set to 10 ppm, and the main search tolerance was set to 20 ppm (other settings were default). Peptide-spectrum matching and protein identification were filtered using a target-bait strategy at 1% FDR. Label-free quantification (LFQ) was selected, with a minimum ratio count of 2. Peptide-spectrum matching scoring and protein identification were filtered using a target-bait strategy at 1% FDR.

[0213] Only proteins quantified in at least 50% of replicates under at least one condition were retained. Normalization was performed by applying RUVIIIC[2], with the most invariant sequence used as a negative control. The optimal k value was determined based on the PCA, RLE, and p-value distribution plots.

[0214] Missing values ​​were filled using the Non-Random Missing Values ​​(MNAR) method. This was achieved by replacing “NA” with a number extracted from a normal distribution whose mean was shifted 1.8 standard deviations to the left of the sample mean and whose width was 0.3.

[0215] Differential expression and enrichment analysis Differential analysis was performed using the R package limma (v. 3.50.1) to compare the differential protein abundance of protein sequences identified in the RhdNeg and RhdPos samples. A protein sequence was considered differentially expressed if the false discovery rate (FDR) was ≤0.05 and the fold change was 2.

[0216] Data Refinement Proteomics data of RhD-specific antibodies were compared with total IgG sorted from single cells. + The antibody sequences obtained from B cells were compared.

[0217] Specifically, RhD-specific antibody sequences were extracted and converted into FASTA files for analysis. Datasets were merged, and IGHM sequences were removed. If a sequence was unique, a new name was assigned to the heavy chain, such as clone-1, clone-2, etc. In cases where cells had identical heavy-light chain pairings, only one cell was included in the analysis, and repetitive sequences in other cells were excluded. Therefore, combining transcriptomic and proteomic sequence information to further assemble and obtain the complete amino acid sequences of the most abundant mAbs in the RhD-specific IgG subset proved to be very robust and efficient.

[0218] This quantitative proteomics method identified the LC and HC regions from RhD. + Specific enrichment in IgG (with RhD) - The method of sequencing RhD peptides (compared to IgG) is novel. Furthermore, these sequences are ranked based on p-values ​​corrected for quantitative enrichment fold changes. This method differs from simple peptide sequencing. Without this quantitative proteomics comparison, the specific RhD sequences listed in this paper could not be identified.

[0219] Example 2: The following method was used to identify antibodies with high affinity.

[0220] Then, the antibody genes identified in Experiment 1 were synthesized and cloned into vectors expressing the γ, κ, or λ constant regions of human antibodies. Figure 2 Based on single-cell NGS data, each antibody variable gene was cloned into a DNA plasmid expressing its native heavy chain constant region and light chain constant region. The paired antibody heavy and light chain expression DNA plasmids were co-transfected into Expi-CHO cells according to the manufacturer's instructions (ThermoFisher Scientific). The antibodies were purified with Protein G agarose and used for further characterization.

[0221] The bioactivity of preferred mAbs (referred to as specific clones throughout this specification) was characterized and analyzed using the techniques described herein.

[0222] RBC preparation Collect RhD + (R0r phenotype: Dce / dce) and RhD - (r'r phenotype: dCe / dce) Blood type O, centrifuged at 200 xg for 10 min. Discard the plasma and add 3 mL of DPBS (+2 mM EDTA) to the cells. Centrifuge the tube at 200 xg for 10 min, discarding the white membrane and supernatant. Wash the cells twice more with DPBS (+2 mM EDTA) and use immediately for experiments.

[0223] To deglycerolize RBCs, incubate the cryovials at 37°C for 2 min, gently invert, and centrifuge at 1500 xg for 5 min. Discard the supernatant except for the last 50 μL of lamellar RBC precipitate. Resuspend the packed RBCs and add 12% NaCl (25 μL, for more than 5 min). Incubate the cells at room temperature for 3 min to equilibrate. Add 237.5 μL of 1.6% NaCl (for more than 5 min), followed by another 0.5 mL of 1.6% NaCl (also for more than 5 min). Transfer the RBCs to a 15 mL sterile conical tube and add 1.25 mL of isotonic wash buffer (0.8% NaCl, 0.2% (11 mM) glucose, pH 7.4) (for more than 5 min). After adding 4 mL of isotonic wash buffer (for more than 8 min), centrifuge the cells at 1500 xg for 5 min. Resuspend the RBC precipitate in 5 mL of buffer (a 3:1 mixture of 1.6% NaCl and isotonic wash buffer) and centrifuge at 1500 x g for 5 min. Finally, resuspend the washed RBCs in DPBS and use immediately or store at 4°C for up to one week.

[0224] RBCs were treated with bromelain to remove surface glycans, which was expected to improve antibody accessibility and thus enhance ADCC. For bromelain treatment, washed RBCs were resuspended in DPBS and treated with bromelain as follows: 2% RBCs in DPBS were mixed with 0.5% bromelain (w / v) (1:2 ratio) and incubated at 37°C for 10 min. Cells were centrifuged at 1500 xg for 5 min and washed twice with DPBS.

[0225] Aggregation test An agglutination assay was performed to evaluate the RhD binding of the candidate antibody. In short, 50 mL of blood type O RhD treated with bromelain was agglutinated. + RBC or RhD - RBCs (1% hematocrit), 25 mL mAb, and 25 mL anti-IgG secondary antibody-AHG chromogenic reagent (BIO-RAD Laboratory #804115) were added to each well of a U-bottom 96-well plate and incubated at room temperature for 90 min. Agglutination was then observed visually.

[0226] To further evaluate the agglutination activity of the candidate mAb against RBCs, serially diluted mAbs were incubated with bromelain-treated and untreated RBCs in the presence of anti-IgG secondary antibody-AHG chromogenic reagent. Plates were incubated at 37°C for 1 hour (using bromelain-treated RBCs) or 2–3 hours (using untreated RBCs). Agglutination was then visually observed and scored. Commercially available (unenriched) polyclonal RhD-pIgG-1 was used as a control in this experiment.

[0227] RBC integration (based on FACS) To assess the binding of mAb to the RhD antigen, flow cytometry analysis was performed. In short, mAb (50 mL) was mixed with 50 mL of blood type O RhD antigen treated with bromelain. + RBC or RhD - RBCs (200,000) were incubated in 96-well U-bottom plates at 37°C for 30 min. Cells were washed with 200 mL of washing buffer (DPBS containing 0.5% BSA and 2 mM EDTA) and then incubated at 1500 mL of water. xg Centrifuge for 2 minutes. Discard the supernatant and add a 1 / 1000 dilution of Fab goat anti-human IgG (H&L) Texas Red (Rockland Antibody and Test #809-1902) to each well. Incubate the plate at room temperature for 30 minutes and then at 1500... xg Centrifuge for 2 minutes. Wash the plate once more with 200 mL of wash buffer and resuspend the cells in 50 mL of wash buffer. Obtain samples on a Fortessa instrument (BD) and analyze using FlowJo v10.6.2. Commercially available (unenriched) polyclonal RhD-pIgG-1 was used as a control in this experiment.

[0228] Competitive binding test between mAb Competitive RBC binding assays were performed to assess whether anti-RhD mAb targets the same or overlapping epitopes, and to what extent they can compete for binding.

[0229] Therefore, the binding of FITC-conjugated anti-RhD mAb to other anti-RhD mAbs was evaluated under conditions of presence and absence. In short, 100,000 bromelain-treated RhD... + RBC (50 μL) and uncoupled mAb (50 μL) were incubated at 37°C for 40 minutes, and then incubated at 1500 °C. xgCentrifuge for 1 minute to remove unbound antibodies. Discard the supernatant and wash RBCs with DPBS. Then, add FITC-conjugated anti-RhD mAb (100 μL) and incubate cells at 37°C for 40 minutes. Wash cells as described above and resuspend in DPBS. Obtain samples on a Fortessa instrument (BD) and analyze using FlowJo v10.6.2. Brad3 and Fog1 mAb were used as positive controls for anti-RhD mAb binding.

[0230] So that approximately 70% of mAb and RhD + The concentration of RBC binding is determined using a FITC-coupled mAb. The concentration of the uncoupled mAb used, where feasible, should be such that it results in at least 90% inhibition in a self-competitive test with the corresponding FITC-coupled mAb.

[0231] THP-1 phagocytosis assay (based on FACS) The THP-1 mononuclear cell line (ATCC) was cultured in RPMI medium with 10% FCS at 37°C and 5% CO2. 10,000 cells were incubated on ice for 30 min with 1 / 40 dilution of mouse anti-human CD64 (FcγRI)-PE Cy7 (BD Biosciences; catalog number 561191), 1 / 10 dilution of mouse anti-human CD32 (FcγRII)-PE (BD Biosciences; catalog number 552884), and 1 / 10 dilution of mouse anti-human CD16 (FcγRIII)-FITC (BD Biosciences; catalog number 555406) ​​to evaluate FcγR expression. Cells were washed with DPBS after centrifugation at 500 xg for 5 min. Samples were obtained on a Fortessa instrument (BD) and analyzed using FlowJo v10.6.2.

[0232] For the phagocytosis assay, 1 μM CellTrace was used in DPBS at 37°C. TM THP-1 monocytes were labeled with Violet (CTV) (Invitrogen; catalog number C34571) for 1 hour with gentle shaking. Cells were washed with RPMI + 10% FCS and centrifuged at 500 xg for 5 min. RPMI + 10% FCS was added to the cells, and the cells were incubated at 37°C for 15 min, followed by washing and resuspending in RPMI + 10% FCS (4 × 10⁻⁶). 5 (cells / mL). 2 × 10 7 One RBC treated with bromelain was resuspended in 1 mL of DPBS and 1 μL of amine-reactive pHrodo was added.TM Red, succinimide ester (ThermoFisher Scientific; catalog number P36600). Cells were incubated at 37°C for 1 hour with gentle shaking, washed with RPMI + 10% FCS, and centrifuged at 1500 xg for 5 min. RBCs were resuspended in RPMI + 10% FCS and incubated at 37°C for 15 min, then washed and resuspended in RPMI + 10% FCS (8 × 10⁻⁶). 6 (cells / mL).

[0233] To detect phagocytosis, different concentrations of antibody (25 μL) were mixed with 2 × 10⁻⁶ mol / L of 2000 mol / L antibody. 5 pHrodo TM RED-marked RhD + 25 μL of cytotoxicity (CTV) and 20,000 CTV THP-1 mononuclear cells (50 μL) were incubated in 96-well U-shaped plates at 37°C for 16 h. The plates were centrifuged at 500 xg for 5 min, then 100 μL of cold RBC lysis buffer was added, and the plates were incubated on ice for 5 min. The plates were washed twice with DPBS, and samples were obtained on a Fortessa instrument (BD) and analyzed using FlowJo v10.6.2. FACS figures are shown in the figure. The phagocytic score was calculated using the formula: (CTV) + / pHrodo red + Cellular percentage (× mean fluorescence intensity) / 1000. Phagocytosis score of the "no Ab" control was subtracted from all samples. Enriched RhD-pIgG-1 was used as a control in this experiment.

[0234] ADCC Trial (Based on FACS) RhD treated with bromelain + RBCs were resuspended in RPMI medium containing 3% human AB serum and 7% FCS at a concentration of 80,000 cells / mL. Then, 25 mL of RhD... + RBCs (20,000 cells / well), 25 mL of purified antibody (100 ng / well or 1 μg / mL in RPMI + 3% human AB serum + 7% FCS), and 50 mL of effector cells (600,000 monocyte-depleted PBMCs / well) were incubated overnight at 37°C. Subsequently, 2 mL of Triton X-100 (ThermoFisherScientific #28314) was added to the control wells (antibody-free control), and the cells were incubated at 37°C for 5 minutes to achieve maximum hemoglobin release. The cells were then incubated at 1500... xgCentrifuge for 2 minutes and collect the supernatant. Use the Human HB (Hemoglobin) ELISA Kit (Elabscience #E-EL-H0415) according to the manufacturer's instructions, employing a 1 / 100 dilution of the supernatant to detect hemoglobin in each well. Calculate the percentage of specific RBC lysis (% ADCC) using the formula: 100 × (Experimental Release - Spontaneous Release) / (Maximum Release - Spontaneous Release). Use enriched RhD-pIgG-1 prepared according to Experiment 2 as a control in these assays.

[0235] NK cell activation (based on FACS) To prepare effector cells, frozen PBMCs were thawed at 37°C, and then NK cells were enriched using a human NK cell isolation kit (Miltenyi Biotec #130-092-657) according to the manufacturer's instructions. 100,000 RhD cells treated with bromelain were... + RBCs (25 μL) were incubated with 100 ng / mL antibody (25 μL) and 100,000 purified NK cells (50 μL) at 37°C for 4 hours, followed by staining for surface or intracellular markers. Commercially available (unenriched) polyclonal RhD-pIgG-1 was used as a control in these assays.

[0236] Cytokine release assay To prepare effector cells, frozen PBMCs were thawed at 37°C, and then NK cells were enriched using a human NK cell isolation kit (Miltenyi Biotec #130-092-657) according to the manufacturer's instructions. 100,000 RhD cells treated with bromelain were... + RBCs (25 μL) were incubated overnight at 37°C with 100 ng / mL antibody (25 μL) and 100,000 purified NK cells (50 μL). The supernatant was then collected, and the presence of cytokines or chemokines was investigated using a Bioplex kit (Bio-rad) according to the manufacturer's instructions. Commercially available (unenriched) polyclonal RhD-pIgG-1 was used as a control in these assays.

[0237] Epitope localization Epitope localization was performed using different experiments.

[0238] Microagglutination assay: 50 mL of bromelain-treated RBCs (1% hematocrit), 25 mL of antibody, and 25 mL of anti-IgG secondary antibody-AHG chromogenic reagent (BIO-RAD Laboratory #804115) were added to each well of a U-bottom 96-well plate and incubated at room temperature for 90 min. Agglutination was then observed visually. The antibody concentrations of the various anti-RhD mAb variants of the present invention used in the assay ranged from 1 to 25 μg / mL.

[0239] The indirect antiglobulin test (IAT) was performed using the Bio-Rad LISS / Coombs kit (BIO-RAD) according to the manufacturer's instructions.

[0240] FACS-based binding assay: Flow cytometry analysis was performed to assess the binding of the antibody (mAb) to the RhD antigen. Briefly, 50 mL of antibody and 50 mL of bromelain-treated RBCs (200,000 cells) were incubated in U-bottom 96-well plates at 37°C for 30 min. Cells were washed with 200 mL of DPBS and incubated at 1500 mL / min. xg Centrifuge for 2 minutes. Discard the supernatant and add a 1 / 1000 dilution of Fab goat anti-human IgG (H&L) Texas Red (Rockland Antibody and Test #809-1902) to each well. Incubate the plate at room temperature for 30 minutes and then at 1500 °C. xg Centrifuge for 2 minutes. Wash the plate again with 200 mL of wash buffer and resuspend the cells in 50 mL of wash buffer. Obtain samples on a Fortessa instrument (BD) and analyze using FlowJov 10.6.2. Commercial (non-enriched) polyclonal RhD-pIgG-1 was used as a control in this experiment.

[0241] Inhibition of anti-RhD mAb binding via polyclonal RhD-pIgG-1 A competitive RBC binding assay was performed to evaluate whether commercially available polyclonal (unenriched) RhD-pIgG-1 could inhibit the binding of anti-RhD mAb. This assay could demonstrate the relative binding affinity and potential epitope overlap between polyclonal RhD-pIgG-1 and anti-RhD mAb.

[0242] Therefore, the binding of FITC-conjugated anti-RhD mAbs was evaluated under conditions of presence and absence of polyclonal IVIg (a non-RhD antibody pool) and RhD-pIgG-1 (derived from an Australian donor). In short, 100,000 RhD mAbs treated with bromelain were used... + RBC (50 μL) was incubated with 5 μg IVIg or RhD-pIgG-1 (50 μL) at 37°C for 30 minutes, and then incubated at 1500 °C. xg Centrifuge for 1 minute to remove unbound antibodies. Discard the supernatant and wash RBCs with DPBS. Then, add serially diluted FITC-conjugated anti-RhD mAb (100 μL) and incubate cells at 37°C for 30 minutes. Wash cells as described above and resuspend in DPBS. Obtain samples on a Fortessa instrument (BD) and analyze using FlowJo v10.6.2. Fog1 mAb was used as a positive control for anti-RhD mAb binding.

[0243] Enrichment of RhD-pIgG1 Commercially available RhD-pIgG consists of a pool of immunoglobulins (antibodies) and exhibits a higher titer of anti-RhD specific IgG compared to titers found in IgG derived from normal plasma. Currently, commercially available clinical products are derived from treatments for hemolytic disease of the newborn (HDN) or targeting RhD. + Plasma from RhD donors that produces anti-RhD antibodies through RBC immunization. However, these formulations contain a mixture of RhD-specific antibodies and non-RhD antibodies.

[0244] In order to directly compare the bioactivity of the mAb of the present invention with that of commercially available materials, the inventors found it necessary to further increase the RhD titer of the commercially available materials to ensure that the RhD component could be detected in the test.

[0245] Therefore, 1.5 mL (625 IU) of commercially available RhD-pIgG-1 (derived from an Australian donor) was diluted with PBS and mixed with 8 mL of compacted RhD. + RBCs (R0r variant) were incubated. The RBCs were then washed with PBS at least six times to remove non-specific binders. To elute the antibodies bound to the RBCs, 16 mL of 50 mM glycine, 0.9% NaCl, and 2 mM EDTA (pH 2.7) were added, the mixture was vortexed for 5 seconds, and incubated on ice for 2 minutes. The RBCs were immediately centrifuged at 2500 xg for 5 minutes, and the supernatant was collected. The eluted pool of RhD-specific antibodies was obtained by neutralizing the acidic pH with 2.1 mL of 1 M Tris and 5.2% NaCl (pH 8).

[0246] To remove potential RBC protein contaminants, further enrichment was achieved by affinity chromatography of protein G agarose followed by replacement of the buffer with PBS.

[0247] Results of Experiment 2 2.1 RhD agglutinated by anti-RhD monoclonal antibody (mAb) + RBC.

[0248] The results showed that among the 180 candidate mAbs tested, 16 mAbs agglutinated with RhD. + RBC, and for RhD - RBCs showed no agglutination activity (Table 2). The agglutination endpoint concentration or agglutination titer refers to the lowest antibody concentration at which visible RBC agglutination occurs. A lower agglutination endpoint concentration indicates higher antibody affinity and potency, because less antibody is required to achieve agglutination.

[0249] Furthermore, we observed that most anti-RhD mAbs were effective against both untreated and bromelain-treated RhD. + When tested on RBCs, their aggregation activity showed no significant difference. These findings indicate that the binding of these mAbs is unaffected by the glycans on the RBC surface and is independent of glycan modification. Figure 4 RhD-pIgG-1 is a polyclonal product containing anti-RhD antibodies derived from Australian blood donors. It is currently available on the market and agglutinates RhD. + RBC, and also RhD - RBCs exhibited mild nonspecific reactivity.

[0250] Table 2. Aggregation activity against RhD mAb.

[0251]

[0252] K: κ light chain; L: λ light chain.

[0253] 2.2 Study the binding of anti-RhD mAb to RBC.

[0254] Anti-RhD mAb showed similarities to RhD. + It exhibits specific binding to RBCs. Notably, clone-30 mAb demonstrates binding with over 75% of RhD. + RBC binding was observed, with seven mAbs (clone 18, clone 27, clone 38, clone 55, clone 65, clone 91, and clone 104) showing binding to 51%–75% of cells. Four mAbs (clone 21a, clone 34, clone 44, and clone 107) showed moderate binding, interacting with 25%–50% of RBCs, while four other mAbs (clone 1a, clone 17, clone 75, and clone 142) showed weak binding, binding to less than 25% of cells (Table 3).

[0255] Table 3. RBC (%) binding of isolated mAbs

[0256] The RBC binding evaluation concentration of mAbs is 1 μg / mL, except for clone 1a-IgG3 (5 μg / mL), clone 17 (5 μg / mL), clone 21a-IgG2 (5 μg / mL), clone 75 (25 μg / mL), clone 142 (5 μg / mL), and commercial RhD-pIgG1 (25 μg / mL). Unless otherwise stated, all mAbs are IgG1. Pool K consists of 16 anti-RhD mAbs with different Fc isotypes. Unbound is indicated by "-". Although commercial RhD-pIgG1 has a high RhD titer, it is not a specifically enriched anti-RhD antibody.

[0257] 2.3 Competitive binding test between mAb Of the mAbs tested, five mAbs (including clone-1a, clone-30, clone-44, clone-65, and clone-104) showed significant RBC binding inhibition against all other mAbs tested, with inhibition rates exceeding 70%. Figure 5A Clones-38, 55, and 91 also exhibited broad inhibitory activity, inhibiting binding to all mAbs, but with slightly reduced inhibition against 3-4 mAbs. On the other hand, the remaining mAbs (including clones-18, 21a, 27, 34, and 107) showed partial inhibition against other mAbs. While they were able to inhibit binding to some mAbs, their inhibitory effects varied and were not as comprehensive as observed in the eight mAbs listed above. Clones-17, 75, and 142 showed the lowest competitive activity. Clone-106 was identified as a non-RhD mAb and showed no inhibition against RhD mAbs, indicating the specificity of this assay. This comprehensive analysis provides valuable insights into the inhibitory potential of these mAbs and their cross-reactivity.

[0258] Of the five mAbs that were able to inhibit RBC binding of all 13 tested mAbs, Clono-104 showed the highest potency, with an inhibitory concentration of 0.5 μg / mL. Figure 5B-C). Following closely, clone-30, at a concentration of 2 μg / mL, exhibited significant inhibitory activity comparable to the Brad3 control anti-RhD mAb. For clone-44, complete inhibition required a concentration of 10 μg / mL, while clone-1a, clone-55, and clone-65 required higher concentrations of 20 μg / mL to achieve complete inhibition. These findings highlight the varying potency of different mAbs in inhibiting RBC binding, with clone-104 and clone-30 exhibiting the strongest inhibitory activity at lower concentrations. Clones-142, clone-17, and clone-75 inhibited binding to fewer than seven other mAbs, with inhibition levels below 50%. This suggests that the epitopes targeted by these mAbs may not significantly overlap with those recognized by mAbs whose binding was not effectively inhibited.

[0259] 2.4 Results of Tabletop Location like Figure 6A As shown, all RhD mAbs (including Brad3, Fog1, and commercially available RhD-pIgG-1) specifically bind to wild-type (WT) RhD variants (R0r, R1R1, and R2R2) but not to rr variants (RhD... - RBC-specific binding. Testing of type 2 weak RhD from different donors revealed distinct binding to clone-17, clone-75, and clone-142. These mAbs also did not bind to type 1 weak RhD. Furthermore, clone-17, clone-75, clone-34, and clone-55 did not bind to type 4 weak RhD. Other tested mAbs showed satisfactory binding to weak RhD variants.

[0260] Most mAbs (clones) showed reactivity to the DVII and DSC2 partial RhD variants, with only two mAbs (clone 27 and clone 142) exhibiting binding to the DVI variant. These mAbs showed different binding to other partial RhD variants, while a few mAbs (clone 30, clone 38, clone 44, and clone 91) consistently showed strong binding to partial RhD variants. Figure 6B As shown, only clone-27 and clone-142 could potentially interact with epitopes 3.1 and 4.1. Clone-142 was the only mAb showing potential reactivity with epitope 15.1, while the other mAbs showed varying results due to different levels of reactivity with the tested RhD variants. Reactivity with epitopes 10.1 and 11.1 could not be determined because RhD variants exposing these epitopes were unavailable. Of the mAbs tested, clone-30, clone-38, clone-44, clone-55, clone-91, and clone-142 showed potential reactivity with all other listed epitopes, while the other mAbs produced varying results.

[0261] 2.5 Inhibition of anti-RhD mAb binding via polyclonal RhD-pIgG-1 like Figure 7A As shown, binding to all mAbs was inhibited by RhD-pIgG-1. Clones-1a, 21a, and 142 were detected to be slightly inhibited by IVIg. Figure 7B The results showed that 44%–79% of mAb binding was inhibited by RhD-pIgG-1, with the lowest inhibition against clone-38 (44%) and the highest inhibition against clone-1a and clone-27 (79%). The significant inhibition of mAb binding by RhD-pIgG-1 suggests that these mAbs target similar or overlapping epitopes on the RhD antigen. The differential inhibition observed among these mAbs indicates differences in epitope specificity and binding strength.

[0262] Evaluation of the activity of 2.6 mAb in mediating RBC clearance via THP-1 monocytes use Figure 8A Flow cytometry analysis using a gating strategy revealed that THP-1 monocytes express CD64 (FcγRI) and CD32 (FcγRII) receptors on their surface, but do not express CD16 (FcγRIII). Figure 8B Except for clone 21a IgG2, all tested anti-RhD mAbs were able to mediate RhD transmission through THP-1 monocytes. + RBC clearance. Most mAbs and RhD-pIgG-1 exhibit a prozone-like effect at concentrations exceeding 100 ng / mL. Figure 8C This indicates that the antibody is in excess relative to the antigen. The mAbs exhibited varying degrees of ADP activity, with six (clone 27, clone 55, clone 65, clone 75, clone 104, and clone 107) showing ADP activity comparable to RhD-pIgG-1. Figure 8D Notably, the subclass conversion from IgG2 to IgG1 in clone 21a increased ADP activity. Figure 8E ).

[0263] These findings indicate that specific anti-RhD mAb can effectively induce monocyte-mediated RhD. + RBC clearance, likely achieved through interaction with Fcγ receptors and subsequent activation of effector functions. Furthermore, preferred embodiments of the invention include the selection of mAbs with IgG1 subclass dominance to enhance ADP activity. The pre-banding effect observed at higher concentrations further underscores the potential for fine-tuning antibody formulations to achieve optimal therapeutic outcomes. The unique properties of these anti-RhD mAbs highlight their potential for development into RhD-related... +The potential of effective treatments for RBC symptoms.

[0264] Evaluation of the activity of 2.7 mAb in RBC clearance mediated by antibody-dependent cytotoxicity (ADCC). Evaluation of ADCC or RBC lysis based on hemoglobin ELISA assay Figure 9A Although the ADCC activity of RhD-pIgG-1 was 48%, the ADCC activity of all tested mAbs was less than 20%. Figure 9B Among the IgG1 mAbs, clones 30 (19.9%), 65 (16.1%), 91 (17.6%), and 107 (15.4%) showed the highest ADCC activity, suggesting that targeting epitopes may influence ADCC activity. Notably, clone 104 (IgG3) exhibited 15.4% RBC cleavage activity; however, conversion to the IgG1 subclass resulted in a decrease to 10.5% (…). Figure 9C Furthermore, the subclass conversion from IgG2 to IgG1 in clone 21 increased ADCC activity by 2.5 times (from 1.8% to 4.6%).

[0265] According to the literature, antibodies derived from plasma donors exhibit different glycosylation patterns compared to normal antibodies, which enhances their ADCC activity. This may be the case with RhD-pIgG-1. However, when mAbs are generated in CHO cells, the glycosylation differs from that of natural anti-RhD antibodies in the donor, reducing their ADCC activity. This suggests that further antibody engineering is needed to achieve comparable ADCC activity.

[0266] 2.8 Study on the mechanism of NK cell clearance of RBCs In assessing NK cell cytokine and chemokine secretion, the mAb of this invention exhibited bioactivity comparable to other commercially available antibodies. The cytokine and chemokine secretion profile of the mAb was very similar to that of commercial RhD-pIgG-1, indicating that the two antibody formulations induced similar immune response patterns. Among the cytokines and chemokines found to be upregulated were RANATS, MIP-1a, MIP-1B, IL-8, IL-9, IL-17, IL-12, IL-1b, IFN-γ, and TNF-α. Figure 10AThese immune mediators play distinct roles in the immune response, including recruiting and activating immune cells to sites of inflammation. While they each play similar roles in inducing RBC death, the combined effects of these immune mediators may promote RBC clearance through phagocytosis and immune cell-mediated cytotoxicity. This enhanced immune response suggests that NK cells are activated upon encountering RBCs opsonized with anti-RhD antibodies, highlighting the complex interactions among these antibodies, NK cells, and RBCs in RhD-associated immune-mediated hemolytic dysfunction.

[0267] In response to anti-RhD mAb, upregulation of intracellular IFN-γ and TNF-α and increased expression of CD107 on the surface of NK cells were observed, consistent with the pattern seen with RhD-pIgG-1. Figure 10B Furthermore, certain anti-RhD mAbs increased the expression of CD69, NKP46, and NKG2D on the surface of NK cells. These findings highlight the similarities between anti-RhD mAbs and commercially available RhD-pIgG-1 in terms of immune activation, as evidenced by their ability to upregulate immune mediators and NK cell activation markers. The collective effect of these immune responses may contribute to RBC clearance in RhD-related hemolytic disorders.

[0268] 2.9 mAb subtypes and germline gene usage study.

[0269] The nucleotide sequences of antibody heavy chain variable genes and light chain variable genes were analyzed using IMGT-V-Quest to identify and recognize their respective germline gene counterparts.

[0270] Of the 16 isolated anti-RhD mAbs, most (13) were classified as IgG1, with one antibody being IgG2 and two antibodies being IgG3 (see Table 4). The major germline gene utilized in these antibodies is IGHV3-33 (…). Figure 11(Table 5) This is consistent with previous studies reporting that the VH3.33 superspecies (including IGHV3-30, IGHV3-30·3, IGHV3-30·5, and IGHV3-33) are the dominant gene regions for anti-RhDmAb (McGowan et al., 2021; Andersen et al., 2007; Dohmen et al., 2006). Nevertheless, the antibodies we isolated also showed the use of other germline genes, such as IGHV1-2, IGHV2-26, IGHV3-21, IGHV3-30, IGHV3-53, IGHV4-34, IGHV4-39, and IGHV4-59. Within the IGHD region, germline genes IGHD1-26, IGHD2-2, IGHD2-21, IGHD3-3, IGHD3-9, IGHD3-10, IGHD3-16, IGHD3-22, IGHD5-12, IGHD5-18, IGHD6-19, and IGHD6-6 were identified. The dominant germline genes in the JH domain were IGHJ4 and IGHJ6, while IGHJ3 and IGHJ5 were less common (Table 5). Notably, antibody clones 1a and 91 share the same germline genes (IGHV3-33, IGHD5-18, and IGHJ4), but functional assays showed they did not respond to RhD. + RBC binding and activity differ.

[0271] The mAbs of this invention exhibit CDRH3 sizes ranging from 15 to 22 amino acids (medium-long length) (Tables 4 and 5), with IGHV somatic hypermutation (SHM) levels ranging from 0.69% to 13.12% (Table 5). CDRH3 size plays a crucial role in antibody binding specificity and affinity; longer CDRH3 regions are generally associated with increased antigen recognition and enhanced potential for binding to target epitopes.

[0272] IGHJ mutations exhibited relatively varying levels, ranging from 0% to 20.83% (Table 5). Based on these findings, SHM levels in the IGHV gene can be categorized as relatively low to moderate. The percentage of IGHV gene mutations indicates the extent to which the antibody sequence changes during the immune response; a higher percentage indicates greater diversity. In this context, the observed mutation levels are in the moderate range, suggesting that the antibody has undergone some degree of mutation, but not to the extent seen in highly mutated antibodies. CDRH3 size also plays a crucial role in antibody binding specificity and affinity; longer CDRH3 regions are generally associated with increased antigen recognition and enhanced potential for binding to target epitopes.

[0273] Of the 16 antibodies analyzed, 10 showed λ light chains, while 6 showed κ light chains (Table 6). The identified germline genes for λ light chains were IGLV1-47, IGLV2-14, IGLV2-23, IGLV7-43, and IGLV1-51, while the germline genes for κ light chains were IGKV1-39 and IGKV2-28. Notably, the major germline genes for both light chain types were IGLV1-47 and IGKV1-39. Figure 12 Regarding mutations, the mutation rate in the IGLV region ranged from 1.04% to 5.38%, while the mutation rate in the J region ranged from 0% to 13.16% (Table 6).

[0274] Compared to the light chain, the isolated antibody exhibited a higher SHM rate and a longer CDR in IGHV. Figure 14A -B). Alignment of the antibody heavy chain variable gene revealed that mAb possesses a unique sequence in its CDRH3 region.

[0275] Table 4: Ig subtypes and CDR size

[0276] Table 5. Germline genes used for the heavy chain in RhD mAb resistance

[0277] Table 6. Germline genes used for the light chain in RhD-resistant mAb

[0278] 2.10 Alphafold modeling.

[0279] Using Alphafold modeling (an AI system that predicts protein 3D structure using amino acid sequences and protein-protein interactions), it can be observed that the antibody heavy chain, especially the CDR H3 region, plays a key role as the main antibody paratopes. Figure 13 Further analyses predicted that the antibody would form hydrogen bonds and salt bridges with the external RhD ring (Table 7). Figure 14A The predicted interacting amino acids on the RhD antigen were shown, while Figure 14B This reveals potential binding sites on the molecule. Asp350 is a common target site in modeling mAbs. Figure 15 ).

[0280] Table 7. Predicted interaction between anti-RhD mAb and RhD antigen.

[0281]

[0282] Experiment 3: Production of monoclonal or multi-cell libraries.

[0283] Now that high-affinity clones have been identified, the methods outlined in this invention can be used to produce monoclonal clones from single-cell libraries, or can be used with multi-cell libraries, as described in Frandsen et al, Biotech Bioeng, 108 (9) 2011 or US20090017017.

[0284] Experiment 4: Fc Modification Introducing point mutations into the antibody Fc region can improve ADCC and antibody-dependent phagocytosis (ADP). Glycosylation of the antibody Fc region (more specifically, fucosylation) can also enhance ADCC by improving the interaction between IgG and the Fcγ receptor (FcγR) on NK cells.

[0285] In the following experiments, the inventors used two anti-RhD antibodies (Brad3 and Fog1) described in the literature and performed Fc mutations, glycoengineering, or both of these modifications in the hope of improving ADCC and ADP function.

[0286] 4.1 Antibody preparation.

[0287] Codon optimization was performed on the heavy chain and light chain variable genes of Brad3 (GenBank: X64149.1 and X64162.1), Fog1 (GenBank: X64150.1 and X64163.1) (both RhD mAb), and VRC03 (GenBank: GU980707.1 and GU980706.1) (non-RhD mAb), followed by synthesis using integrated DNA technologies. To construct the IgG1 variant, the synthesized heavy chain and light chain variable genes were used... AgeI / Sall-HF and AgeI / BsiWi-HF Digested and cloned into human γ1 constant region (heavy chain IgG1) and κ constant region (light chain) expression vectors. The IgG3 variant was constructed by: synthesizing the human γ3 (IgG3) constant region (via Integrated DNA Technologies Inc.), and then... Sall-HF / BamHI-HF Digestion and replacement of the IgG1 constant gene in the heavy chain expression vector.

[0288] To create antibody Fc mutant variants (IgG1-GASDALIE and IgG1-GRLR), the heavy chain γ1 gene (IgG1) was mutated to introduce the following mutations: G236A, S239D, A330L, and I332E (IgG1-GASDALIE antibody) and G236R and L328 (IgG1-GRLR antibody). Mutations in the heavy chain Fc gene were performed using overlap extension PCR. Three separate initial PCR reactions were performed with unique oligonucleotides. The products of the three PCR reactions were electrophoresed separately on 2% agarose gels and extracted and purified using Nucleospin gels and a PCR purification kit. The purified PCR products were mixed in a 1:1:1 ratio and subjected to overlap extension PCR to form a continuous Fc sequence. PCR was performed as described above, except that no oligonucleotides were added to the mixture. The final PCR products were subjected to agarose gel electrophoresis, extraction, and purification. Sall-HF / BamHI-HF The purified mutant Fc gene was digested and replaced with the constant IgG1γ1 gene in the heavy chain expression vector.

[0289] Antibody expression vectors containing heavy chain variable and constant (IgG1, IgG1-GASDALIE, IgG1-GRLR, and IgG3) genes and light chain variable and constant (κ) genes were co-transfected into Expi-Chinese hamster ovary (Expi-CHO) cells using the Expi-CHO Expression System Kit. The antibody-containing supernatant was harvested 4–5 days post-transfection and filtered using a 0.22 mm filter. The antibody was purified using Protein G Agarose Fast Flow, and then the buffer was exchanged for PBS using an Amicon 50 kDa Millipore rotating membrane before sterilization.

[0290] To inhibit fucosylation and produce an IgG1-afucosylated (IgG1-Afuc) antibody variant, heavy chain expression plasmids (IgG1 and IgG1-GASDALIE) and light chain expression plasmid (κ) were co-transfected into ExpiCHO cells in the presence of 50 μM fucostatin. The antibodies were harvested and purified as described above.

[0291] The purified antibodies were electrophoresed on NuPage 4%–12% Bis-Tris gels and incubated with anti-human (H+L) antibodies conjugated with goat horseradish peroxidase (HRP), or with biotinylated lectins followed by avidin HRP. Western blotting confirmed reduced fucosylation in the purified mAbs using IgG1-defucosylated mAbs compared to IgG1 mAbs.

[0292] 4.2 Agglutination of RhD by anti-RhD antibody variants + RBC.

[0293] Type O blood red blood cells carrying the RhD antigen (R0r) agglutinate in the presence of all Brad3 and Fog1 variants, but not in the presence of the VRCO3 variant. - Neither type O blood RBCs (r'r) nor the antibody agglutinated, confirming the specificity of Brad3 and Fog1 for the RhD antigen. Agglutination is a highly sensitive assay, and the agglutination endpoint concentration for both Brad3 and Fog1 variants was 5 ng / mL, indicating that the agglutination activity was maintained in the engineered anti-RhD mAb.

[0294] 4.3 Engineered antibodies maintain RBC binding.

[0295] To confirm that the binding of the Fc-engineered antibody to the RhD antigen was maintained, flow cytometry was performed. Figure 16 As shown, modifications to the Fc region of anti-RhD antibodies do not affect RhD antigen binding. None of the antibody variants showed binding to RhD. - RBC binding. It has been confirmed that the VRC03 antibody does not bind to R0r RhD. + RBC.

[0296] 4.4 Glycoengineering and Fc mutations improved NK cell activation and RBC clearance via ADCC. Figure 17 A illustrates a method for evaluating NK cell activation. Figure 17 B demonstrates the effect of these modifications on NK cell activation. Although some differences were observed among the variants, the GASDALIE Fc variant and the IgG1 defucosylated variant, or variants with both modifications, showed no significant difference in enhancing NK cell activation. Consistent results were obtained in ADCC assays, indicating that these antibody modifications improved ADCC compared to the WT variant and showed activity comparable to enriched RhD-pIgG-1. Figure 17 C).

[0297] 4.5. Fc modification maintains ADP activity against RhD mAb.

[0298] like Figure 18 As shown in Figure A, anti-RhD mAb mediates RhD. + Phagocytosis of RBCs was observed, but this activity was not acquired in the control mAb (non-RhD mAb). Phagocytosis by THP-1 monocytes indicated that Brad3 and Fog1 mAb variants mediated RhD-targeting activity. + RhD-specific phagocytic activity of RBC (R0r phenotype) Figure 18 A), while RhD - RBCs (r'r phenotype) failed to mediate any response in the presence of the antibody (data not shown). Fc-modified mAbs also retained ADP activity. Figure 20 D). The mAb variant showed ADP activity comparable to enriched RhD-pIgG-1 ( Figure 18 B).

[0299] Experiment 5: Comparison of the bioactivity of mAbs generated by the method of the present invention with different commercially available hyperimmune RhD IgGs.

[0300] In previous experiments, the inventors used commercially available anti-RhD-pIgG derived from Australian donors. Subsequent experiments repeated some of the previous studies using different commercial products (this time from a US donor). Using hyperimmune products from different sources helps demonstrate the robustness of mAb selection, as different ethnicities may produce antigen profiles with subtle differences, potentially resulting in slightly different antigen-binding fragments, which could then influence the efficacy and bioactivity of the selected mAb.

[0301] Anti-RhD antibodies were enriched from commercially available anti-Rhd-pIgG (RhD-pIgG-2) derived from a US donor.

[0302] In order to directly compare the bioactivity of the mAb of the present invention with that of the commercial material, the inventors again found it necessary to further increase the RhD titer of the commercial material to ensure that the RhD component was detectable in the test.

[0303] Therefore, 2 mL (1500 IU) of commercial RhD-pIgG-2 was diluted with PBS and mixed with 8 mL of compacted RhD. + RBCs (R0r variant) were incubated. The RBCs were then washed with PBS at least six times to remove non-specific conjugates. To elute the antibody binding to the RBCs, 16 mL of 50 mM glycine, 0.9% NaCl, and 2 mM EDTA (pH 2.7) was added, the mixture was vortexed for 5 seconds, and incubated on ice for 2 minutes. The RBCs were immediately centrifuged at 2500 xg for 5 minutes, the supernatant was collected, and neutralized by adding 2.1 mL of 1 M Tris, 5.2% NaCl (pH 8). To remove potential RBC protein contaminants, further enrichment was achieved using affinity chromatography with Protein G agarose. This enriched sample was used as a baseline sample for subsequent experiments.

[0304] Experiment 6: Generating mAb pools. Different combinations of mAbs according to the present invention.

[0305] Different mAbs were mixed to provide different combinations, demonstrating the breadth of the invention. By mixing several different clones (variants) of the mAbs of the invention, the final composition can preferably provide a wider range of reactivity and functional diversity. These pools or combinations may more closely resemble the complex antibody mixtures found in commercial polyclonal RhD-pIgG products. Thus, antibody combinations or antibody pools containing 3, 4, or 16 mAbs were generated (Table 8) and evaluated in agglutination assays (Table 9).

[0306] As detailed in Experiment 4, the Fc regions of clones 27, 30, and 65 mAb were engineered with GASDALIE mutations. Subsequently, condensation (Table 9), ADCC ( Figure 19 AB) and ADP Figure 19 The pools of wild-type and GASDALIE mutant variants of these mAbs were evaluated in a CD assay. GASDALIE engineering preserved the aggregation activity of the mAbs (Table 9). Compared with wild-type mAbs and wild-type pools (pool I), individual GASDALIE mAbs and GASDALIE pools (pool J) showed improved ADCC activity, particularly when using normal (untreated) RBCs. Figure 19 AB). When using RBCs treated with bromelain, the ADCC activities of mAb and cytoplasm were comparable to those of RhD-pIgG1 (AB). Figure 19 A). However, when using untreated RBCs, GASDALIE mAb and GASDALIE pool (pool J) exhibited higher ADCC activity than RhD-pIgG1 ( Figure 19 B). Compared to the wild-type form, the GASDALIE variant retained ADP activity, and the ADP activity of mAb and pool was comparable to the ADCC activity of RhD-pIgG1 (B). Figure 19 CD).

[0307] exist Figure 19 The study included non-RhD allotype control mAbs, and signals above the background levels of these negative control samples were defined as positive ADCC / ADP signals. Background ADCC / ADP activity from the negative control mAbs was subtracted in all experiments.

[0308] The combination or pool of mAb showed ADCC activity against RBCs treated with bromelain. Figure 20 A), showed ADP activity in both treated and untreated RBCs (A), Figure 20 CD), and the results were comparable to those of two commercial enrichment products, RhD-pIgG-1 and RhD-pIgG-2. Although no pool showed improvement against untreated RhD... +RBC ADCC activity, but pool J (which contains GASDALIE mAb) showed stronger activity than RhD-pIgG-1 and RhD-pIgG-2. Figure 20 B). Pool J for unprocessed RhD + This enhanced RBC activity highlights the efficacy of GASDALIE-engineered mAbs, providing a potentially more effective treatment option for targeting untreated RBCs. The superior performance of this study underscores the innovativeness of this approach, establishing a novel and innovative method for enhancing ADCC activity of anti-RhD mAbs, which may have important clinical and therapeutic applications.

[0309] exist Figure 20 The study included a negative control pool (containing non-RhD isotype control mAbs: one IgG1 mAb, one IgG2 mAb, and one IgG3 mAb). Signals above the background of the negative control pool were defined as positive ADCC signals. Background ADCC activity from the negative control pool was subtracted in each experiment. Each column represents the mean ADCC percentage, and the error column represents the standard error of the mean of two independent experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett's multiple comparison test (GraphPadPrism v9) to compare the activity of each antibody against RhD-pIgG-1. ****: P < 0.0001.

[0310] Table 8: Composition of the pool

[0311] Except for clone-1a, which is IgG3, all other mAbs are IgG1.

[0312] Table 9: Aggregation activity of mAb pools and engineered mAbs.

[0313]

[0314] Experiment 7: Study on the minimum antibody fragment required for RBC binding RhD treated with bromelain + RBCs were exposed to anti-RhD mAb, including the full-length Brad3_ fragment, the Brad3_F(ab')2 fragment, and the Brad3_Fab fragment. Binding to the antibody fragments was then evaluated using FACS.

[0315] like Figure 21 As shown, the results indicate that all segments of Brad3 (including Brad3_full length, Brad3_F(ab')2, and Brad3_Fab) exhibit characteristics consistent with RhD. +RBC binding ability. Although the full-length antibody and the F(ab')2 fragment showed similar binding activity, the Fab fragment showed slightly lower binding activity.

Claims

1. An isolated or recombinant antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD), wherein said antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising: a complementarity-determining region (CDR) H1 comprising a sequence as shown in any of the following: SEQ ID NO: 11, SEQ ID NO: 43, SEQ ID NO: 75, SEQ ID NO: 107, SEQ ID NO: 139, SEQ ID NO: 171, SEQ ID NO: 203, SEQ ID NO: 235, SEQ ID NO: 267, SEQ ID NO: 299, SEQ ID NO: 331, SEQ ID NO: 363, SEQ ID NO: 395, SEQ ID NO: 427, SEQ ID NO: 459, SEQ ID NO: 491; and a CDR H2 comprising a sequence as shown in any of the following: SEQ ID NO: 13, SEQ ID NO: 45, SEQ ID NO: 7 ...07, SEQ ID NO: 107, SEQ ID NO: 107, SEQ ID NO: 107, SEQ ID NO 109, SEQ ID NO: 141, SEQ ID NO: 173, SEQ ID NO: 205, SEQ ID NO: 237, SEQ ID NO: 269, SEQ ID NO: 301, SEQ ID NO: 333, SEQ ID NO: 365, SEQ ID NO: 397, SEQ ID NO: 429, SEQ ID NO: 461, SEQ ID NO: 493; and CDR H3, comprising a sequence as shown in any of the following: SEQ ID NO: 15, SEQ ID NO: 47, SEQ ID NO: 79, SEQ ID NO: 111, SEQ ID NO: 143, SEQ ID NO: 175, SEQ ID NO: 207, SEQ ID NO: 239, SEQ ID NO: 271, SEQ ID NO: 303, SEQ ID NO: 335, SEQ ID NO: 367, SEQ ID NO: 399, SEQ ID NO: 431, SEQ ID NO: 463, SEQ ID NO:

495.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 11, the CDR H2 sequence as shown in any one of SEQ ID NO: 13, and the CDR H3 sequence as shown in SEQ ID NO:

15.

3. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 43, the CDR H2 sequence as shown in SEQ ID NO: 45, and the CDR H3 sequence as shown in SEQ ID NO:

47.

4. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 75, the CDR H2 sequence as shown in SEQ ID NO: 77, and the CDR H3 sequence as shown in SEQ ID NO:

79.

5. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 107, the CDR H2 sequence as shown in SEQ ID NO: 109, and the CDR H3 sequence as shown in SEQ ID NO:

111.

6. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 139, the CDR H2 sequence as shown in SEQ ID NO: 141, and the CDR H3 sequence as shown in SEQ ID NO:

143.

7. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 171, the CDR H2 sequence as shown in SEQ ID NO: 173, and the CDR H3 sequence as shown in SEQ ID NO:

175.

8. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 203, the CDR H2 sequence as shown in SEQ ID NO: 205, and the CDR H3 sequence as shown in SEQ ID NO:

207.

9. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 235, the CDR H2 sequence as shown in SEQ ID NO: 237, and the CDR H3 sequence as shown in SEQ ID NO:

239.

10. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 267, the CDR H2 sequence as shown in SEQ ID NO: 269, and the CDR H3 sequence as shown in SEQ ID NO:

271.

11. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 299, the CDR H2 sequence as shown in SEQ ID NO: 301, and the CDR H3 sequence as shown in SEQ ID NO:

303.

12. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 331, the CDR H2 sequence as shown in SEQ ID NO: 333, and the CDR H3 sequence as shown in SEQ ID NO:

335.

13. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 363, the CDR H2 sequence as shown in SEQ ID NO: 365, and the CDR H3 sequence as shown in SEQ ID NO:

367.

14. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 395, the CDR H2 sequence as shown in SEQ ID NO: 397, and the CDR H3 sequence as shown in SEQ ID NO:

399.

15. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 427, the CDR H2 sequence as shown in SEQ ID NO: 429, and the CDR H3 sequence as shown in SEQ ID NO:

431.

16. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 459, the CDR H2 sequence as shown in SEQ ID NO: 461, and the CDR H3 sequence as shown in SEQ ID NO:

463.

17. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment comprises the CDR H1 sequence as shown in SEQ ID NO: 491, the CDR H2 sequence as shown in SEQ ID NO: 493, and the CDR H3 sequence as shown in SEQ ID NO:

495.

18. The antibody or antigen-binding fragment of any one of claims 1 to 17, wherein the antibody or antigen-binding fragment comprises a light chain variable region (VL), comprising: CDR L1, comprising a sequence as shown in any one of the following: SEQ ID NO:27, SEQ ID NO:59, SEQ ID NO:91, SEQ ID NO:123, SEQ ID NO:155, SEQ ID NO:187, SEQ ID NO:219, SEQ ID NO:251, SEQ ID NO:283, SEQ ID NO:315, SEQ ID NO:347, SEQ ID NO:379, SEQ ID NO:411, SEQ ID NO:443, SEQ ID NO:475, SEQ ID NO:507; CDR L2, comprising a sequence as shown in any one of the following: SEQ ID NO:29, SEQ ID NO:61, SEQ ID NO:93, SEQ ID NO:125, SEQ ID NO:157, SEQ ID NO:507; ... SEQ ID NO: 189, SEQ ID NO: 221, SEQ ID NO: 253, SEQ ID NO: 285, SEQ ID NO: 317, SEQ ID NO: 349, SEQ ID NO: 381, SEQ ID NO: 413, SEQ ID NO: 445, SEQ ID NO: 477, SEQ ID NO: 509; and CDR L3, comprising a sequence as shown in any of the following: SEQ ID NO: 31, SEQ ID NO: 63, SEQ ID NO: 95, SEQ ID NO: 127, SEQ ID NO: 159, SEQ ID NO: 191, SEQ ID NO: 223, SEQ ID NO: 255, SEQ ID NO: 287, SEQ ID NO: 319, SEQ ID NO: 351, SEQ ID NO: 383, SEQ ID NO: 415, SEQ ID NO: 447, SEQ ID NO: 479, SEQ ID NO:

511.

19. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 27, a CDR L2 sequence as shown in SEQ ID NO: 29, and a CDR L3 sequence as shown in SEQ ID NO:

31.

20. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 59, a CDR L2 sequence as shown in SEQ ID NO: 61, and a CDR L3 sequence as shown in SEQ ID NO:

63.

21. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 91, a CDR L2 sequence as shown in SEQ ID NO: 93, and a CDR L3 sequence as shown in SEQ ID NO:

95.

22. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 123, a CDR L2 sequence as shown in SEQ ID NO: 125, and a CDR L3 sequence as shown in SEQ ID NO:

127.

23. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 155, a CDR L2 sequence as shown in SEQ ID NO: 157, and a CDR L3 sequence as shown in SEQ ID NO:

159.

24. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises VL, which comprises a CDR L1 as shown in SEQ ID NO: 187, a CDR L2 sequence as shown in SEQ ID NO: 189, and a CDR L3 as shown in SEQ ID NO:

191.

25. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 219, a CDR L2 sequence as shown in SEQ ID NO: 221, and a CDR L3 sequence as shown in SEQ ID NO:

223.

26. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 251, a CDR L2 sequence as shown in SEQ ID NO: 253, and a CDR L3 sequence as shown in SEQ ID NO:

255.

27. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 283, a CDR L2 sequence as shown in SEQ ID NO: 285, and a CDR L3 sequence as shown in SEQ ID NO:

287.

28. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 315, a CDR L2 sequence as shown in SEQ ID NO: 317, and a CDR L3 sequence as shown in SEQ ID NO:

319.

29. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 347, a CDR L2 sequence as shown in SEQ ID NO: 349, and a CDR L3 sequence as shown in SEQ ID NO:

351.

30. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 379, a CDR L2 sequence as shown in SEQ ID NO: 381, and a CDR L3 sequence as shown in SEQ ID NO:

383.

31. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 411, a CDR L2 sequence as shown in SEQ ID NO: 413, and a CDR L3 sequence as shown in SEQ ID NO:

415.

32. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 443, a CDR L2 sequence as shown in SEQ ID NO: 445, and a CDR L3 sequence as shown in SEQ ID NO:

447.

33. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 475, a CDR L2 sequence as shown in SEQ ID NO: 477, and a CDR L3 sequence as shown in SEQ ID NO:

479.

34. The antibody or antigen-binding fragment of any one of claims 1 to 18, wherein the antibody or antigen-binding fragment comprises a VL, which comprises a CDR L1 sequence as shown in SEQ ID NO: 507, a CDR L2 sequence as shown in SEQ ID NO: 509, and a CDR L3 sequence as shown in SEQ ID NO:

511.

35. The antibody or antigen-binding fragment of any one of claims 1 to 34, wherein the VH comprises an amino acid sequence having at least 70% sequence identity with the sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 41, SEQ ID NO: 73, SEQ ID NO: 105, SEQ ID NO: 137, SEQ ID NO: 169, SEQ ID NO: 201, SEQ ID NO: 233, SEQ ID NO: 265, SEQ ID NO: 297, SEQ ID NO: 329, SEQ ID NO: 361, SEQ ID NO: 393, SEQ ID NO: 425, SEQ ID NO: 457, and SEQ ID NO:

489.

36. The antibody or antigen-binding fragment of any one of claims 1 to 35, wherein the VL comprises an amino acid sequence having at least 70% sequence identity with the sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 57, SEQ ID NO: 89, SEQ ID NO: 121, SEQ ID NO: 153, SEQ ID NO: 185, SEQ ID NO: 217, SEQ ID NO: 249, SEQ ID NO: 281, SEQ ID NO: 313, SEQ ID NO: 345, SEQ ID NO: 377, SEQ ID NO: 409, SEQ ID NO: 441, SEQ ID NO: 473, and SEQ ID NO:

505.

37. An antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD), wherein the antibody or antigen-binding fragment thereof comprises: a) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 11; CDR H2, comprising the sequence shown in SEQ ID NO: 13; and CDRH3, comprising the sequence shown in SEQ ID NO: 15; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 27; CDR L2, comprising the sequence shown in SEQ ID NO: 29; and CDR L3, comprising the sequence shown in SEQ ID NO: 31; b) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 43; CDR H2, comprising the sequence shown in SEQ ID NO: 45; and CDR H3, comprising the sequence shown in SEQ ID NO: 47; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 59; CDR L2, comprising the sequence shown in SEQ ID NO: 61; and CDR L3, comprising the sequence shown in SEQ ID NO: 63; c) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 75; a CDR H2, comprising the sequence shown in SEQ ID NO: 77; and a CDR H3, comprising the sequence shown in SEQ ID NO: 79; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 91; a CDR L2, comprising the sequence shown in SEQ ID NO: 93; and a CDR L3, comprising the sequence shown in SEQ ID NO: 95; d) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 107; a CDR H2, comprising the sequence shown in SEQ ID NO: 109; and a CDR H3, comprising the sequence shown in SEQ ID NO: 111; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 123; a CDR L2, comprising the sequence shown in SEQ ID NO: 125; and a CDR L3, comprising the sequence shown in SEQ ID NO: 127; e) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 139; CDR H2, comprising the sequence shown in SEQ ID NO: 141; and CDR H3, comprising the sequence shown in SEQ ID NO: 143; wherein the light chain variable region (VL) comprises: CDR1, comprising the sequence shown in SEQ ID NO: 155; CDR L2, comprising the sequence shown in SEQ ID NO: 157; and CDR L3, comprising the sequence shown in SEQ ID NO: 159; f) Heavy chain variable regions (VH) and light chain variable regions (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 171; a CDR H2, comprising the sequence shown in SEQ ID NO: 173; and a CDR H3, comprising the sequence shown in SEQ ID NO: 175; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 187; a CDR L2, comprising the sequence shown in SEQ ID NO: 189; and a CDR L3, comprising the sequence shown in SEQ ID NO: 191; g) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 203; CDR H2, comprising the sequence shown in SEQ ID NO: 205; and CDR H3, comprising the sequence shown in SEQ ID NO: 207; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 219; CDR L2, comprising the sequence shown in SEQ ID NO: 221; and CDR L3, comprising the sequence shown in SEQ ID NO: 223; h) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 235; CDR2, comprising the sequence shown in SEQ ID NO: 237; and CDR3, comprising the sequence shown in SEQ ID NO: 239; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 251; CDR L2, comprising the sequence shown in SEQ ID NO: 253; and CDR L3, comprising the sequence shown in SEQ ID NO: 255; i) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 267; CDR H2, comprising the sequence shown in SEQ ID NO: 269; and CDR 3, comprising the sequence shown in SEQ ID NO: 271; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 283; CDR L2, comprising the sequence shown in SEQ ID NO: 285; and CDR L3, comprising the sequence shown in SEQ ID NO: 287; j) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 299; CDR H2, comprising the sequence shown in SEQ ID NO: 301; and CDR H3, comprising the sequence shown in SEQ ID NO: 303; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 315; CDR L2, comprising the sequence shown in SEQ ID NO: 317; and CDR L3, comprising the sequence shown in SEQ ID NO: 319; k) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 331; a CDR H2, comprising the sequence shown in SEQ ID NO: 333; and a CDR H3, comprising the sequence shown in SEQ ID NO: 335; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 347; a CDR L2, comprising the sequence shown in SEQ ID NO: 349; and a CDR L3, comprising the sequence shown in SEQ ID NO: 351; l) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 363; CDR H2, comprising the sequence shown in SEQ ID NO: 365; and CDR H3, comprising the sequence shown in SEQ ID NO: 367; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 379; CDR L2, comprising the sequence shown in SEQ ID NO: 381; and CDR L3, comprising the sequence shown in SEQ ID NO: 383; m) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: a complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 395; a CDR H2, comprising the sequence shown in SEQ ID NO: 397; and a CDR H3, comprising the sequence shown in SEQ ID NO: 399; wherein the light chain variable region (VL) comprises: a CDR L1, comprising the sequence shown in SEQ ID NO: 411; a CDR L2, comprising the sequence shown in SEQ ID NO: 413; and a CDR L3, comprising the sequence shown in SEQ ID NO: 415; n) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 427; CDR H2, comprising the sequence shown in SEQ ID NO: 429; and CDR H3, comprising the sequence shown in SEQ ID NO: 431; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 443; CDR L2, comprising the sequence shown in SEQ ID NO: 445; and CDR L3, comprising the sequence shown in SEQ ID NO: 447; o) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 459; CDR H2, comprising the sequence shown in SEQ ID NO: 461; and CDR H3, comprising the sequence shown in SEQ ID NO: 463; the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 475; CDR L2, comprising the sequence shown in SEQ ID NO: 477; and CDR L3, comprising the sequence shown in SEQ ID NO: 479; or p) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) comprises: complementarity determination region (CDR) H1, comprising the sequence shown in SEQ ID NO: 491; CDR H2, comprising the sequence shown in SEQ ID NO: 493; and CDR H3, comprising the sequence shown in SEQ ID NO: 495; wherein the light chain variable region (VL) comprises: CDR L1, comprising the sequence shown in SEQ ID NO: 507; CDR L2, comprising the sequence shown in SEQ ID NO: 509; and CDR L3, comprising the sequence shown in SEQ ID NO:

511.

38. An antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD), wherein the antibody or antigen-binding fragment thereof comprises: a) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 9; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 25; b) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 41; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 57; c) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 73; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in sequence SEQ ID NO: 89; d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 105; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 121; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 137; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 153; f) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

185. g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 201; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

217. h) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 233; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

249. i) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

281. j) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 297; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 313; k) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 345; l) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 361; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

377. m) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 393; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 409; n) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

441. o) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 457; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 473; or p) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3, as shown in SEQ ID NO: 489; and the light chain variable region (VL) containing CDRs L1, L2 and L3, as shown in SEQ ID NO:

505.

39. The antibody or antigen-binding fragment of claim 37 or claim 38, wherein the VH comprises an amino acid sequence having at least 70% sequence identity with the sequence shown in any one of SEQ ID NO: 9, SEQ ID NO: 41, SEQ ID NO: 73, SEQ ID NO: 105, SEQ ID NO: 137, SEQ ID NO: 169, SEQ ID NO: 201, SEQ ID NO: 233, SEQ ID NO: 265, SEQ ID NO: 297, SEQ ID NO: 329, SEQ ID NO: 361, SEQ ID NO: 393, SEQ ID NO: 425, SEQ ID NO: 457, and SEQ ID NO:

489.

40. The antibody or antigen-binding fragment of any one of claims 37 to 39, wherein the VL comprises an amino acid sequence having at least 70% sequence identity with the sequence shown in any one of SEQ ID NO: 25, SEQ ID NO: 57, SEQ ID NO: 89, SEQ ID NO: 121, SEQ ID NO: 153, SEQ ID NO: 185, SEQ ID NO: 217, SEQ ID NO: 249, SEQ ID NO: 281, SEQ ID NO: 313, SEQ ID NO: 345, SEQ ID NO: 377, SEQ ID NO: 409, SEQ ID NO: 441, SEQ ID NO: 473, and SEQ ID NO:

505.

41. An antibody or antigen-binding fragment thereof capable of specifically binding to rhesus monkey D (RhD), wherein the antibody or antigen-binding fragment thereof comprises: a) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 9, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 25; b) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 41 and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 57; c) A heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 73, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 89; d) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 105 and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 121; e) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 137 and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 153; f) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 169, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 185; g) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 201, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 217; h) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 233, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 249; i) The heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 265, and the light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 281; j) Containing a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 297, and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 313; k) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 329, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 345; l) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 361, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 377; m) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 393, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 409; n) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 425, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO: 441; o) comprising a heavy chain variable region (VH) of the sequence shown in SEQ ID NO: 457, and a light chain variable region (VL) of the sequence shown in SEQ ID NO: 473; or p) includes a heavy chain variable region (VH) containing the sequence shown in SEQ ID NO: 489, and a light chain variable region (VL) containing the sequence shown in SEQ ID NO:

505.

42. The antibody or antigen-binding fragment according to any one of claims 1 to 41, wherein, The antibody or its antigen-binding fragment can deplete or eliminate RhD+ red blood cells, but cannot deplete or eliminate RhD-negative (RhD-) red blood cells.

43. The antibody or binding fragment according to any one of claims 1 to 42, wherein, The antibody or binding fragment belongs to IgG subclass 1 and / or IgG subclass 3.

44. A composition comprising one or more antibody or antigen-binding fragments as defined in any one of claims 1 to 43.

45. A composition comprising a combination of three or more antibodies or antigen-binding fragments thereof capable of specifically binding to rhesus monkey D (RhD), wherein said three or more antibodies or antigen-binding fragments are selected from antibodies or antigen-binding fragments thereof comprising: a) Heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 137; and light chain variable region (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 153; b) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 169; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

185. c) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 233; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 249; d) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 265; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO: 281; e) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 329; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

345. f) Heavy chain variable regions (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3 as shown in SEQ ID NO: 393; and light chain variable regions (VL) containing CDRs L1, L2 and L3 as shown in SEQ ID NO:

409. g) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2, and H3, as shown in SEQ ID NO: 425; and the light chain variable region (VL) containing CDRs L1, L2, and L3, as shown in SEQ ID NO: 444; or h) The heavy chain variable region (VH) containing complementarity-determining regions (CDRs) H1, H2 and H3, as shown in SEQ ID NO: 489; and the light chain variable region (VL) containing CDRs L1, L2 and L3, as shown in SEQ ID NO:

505.

46. ​​The composition of claim 44 or 45, wherein it comprises a pharmaceutically acceptable carrier.

47. A method for treating or preventing allogeneic immunization of Rh-negative subjects in rhesus monkeys, comprising administering to an Rh-negative subject an effective amount of any one of claims 1 to 46 of an anti-Rd(D) antibody or antigen-binding fragment, or a combination thereof, or any one of claims 44 to 46 of an Rh-negative subject.

48. The method of claim 47, wherein the subject is pregnant.

49. The method of claim 47, wherein the method prevents hemolytic diseases of the fetus and newborn.

50. Use of the antibody or antigen-binding fragment, or combination thereof, as described in any one of claims 1 to 45, in the preparation of a medicament for the treatment or prevention of hemolytic diseases in fetuses and newborns.

51. The antibody or antigen-binding fragment, or combination thereof, or the composition of any one of claims 1 to 45, for the treatment or prevention of allogeneic immunity in Rh-negative subjects in rhesus monkeys.

52. A method for analyzing the most abundant antibodies binding to an antigen (RhD) in a polyclonal (pAb) population, the method comprising: a) Proteomic analysis of a mixture of RhD-specific IgG in plasma to provide proteomic sequence information: i. Providing donor blood plasma; ii. Affinity purification of the total IgG mixture from the plasma; iii. Through communication with RhD + RBC or RhD - RBC incubation was used to separate RhD-specific IgG subsets and non-RhD-specific IgG subsets, respectively. iv. Using one or more proteases, the RhD-specific IgG subsets and non-RhD-specific IgG subsets are independently fragmented into short peptides with overlapping sequences; v. To identify the sequence of each peptide by analyzing fragmented peptides using mass spectrometry; vi. Assemble the short peptides into longer peptides belonging to a single antibody, optionally using computational software; exclude antibodies derived from non-RhD-specific subsets and do not perform further analysis; b) BCR sequencing: i. Providing B cells isolated from the blood of the donor; ii. Extract RNA from the B cells; iii. Sequencing the RNA using NGS to provide transcriptomics sequence information; c) Combine the transcriptomics sequence information with the proteomics sequence information to further assemble and obtain the complete amino acid sequence of the most abundant mAb in the RhD-specific IgG subset.

53. The method of claim 52, wherein, Step (c) further includes: sorting the sequence by fold changes in enrichment based on quantified values ​​of the RhD-specific IgG subsets to identify the most abundant mAbs that are specifically enriched in both the LC and HC regions.

54. A method for analyzing the most abundant antibodies binding to an antigen (RhD) in a polyclonal (pAb) population, the method comprising: a) Perform proteomic analysis on a mixture of RhD-specific IgG in plasma to provide proteomic sequence information; b) BCR sequencing; c) Combine the transcriptomics sequence information with the proteomics sequence information to further assemble and obtain the complete amino acid sequence of the most abundant mAb in the RhD-specific IgG subset; d) Further select mAb combinations from the most abundant mAbs, wherein each mAb contains a different gene and / or amino acid sequence, and each mAb contains the IgG1 subclass heavy chain variable region (VH).

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