Testosterone binding sequences

Abstract

Disclosed herein are antibodies and antigen-binding fragments that bind specifically to testosterone. The antibodies and antigen-binding fragments may be suitable for therapeutic and diagnostic use in diseases or disorders associated with abnormal testosterone levels.

Description

TESTOSTERONE BINDING SEQUENCES

[0001] This application claims the benefit of priority to Indian Provisional Patent Application No. 202441043701, filed on June 5, 2024; and U.S. Provisional Patent Application No. 63 / 709,233, filed on October 18, 2024. The contents of each of these applications are hereby incorporated by reference in their entirety.

[0002] The present disclosure relates to antibodies and antibody engineering technology. More particularly, the present disclosure relates to antibody and antigen-binding fragments that are specific for testosterone, as well as their use in immunodiagnostics and immunotherapy.

[0003] Testosterone, an essential steroid hormone, is synthesized primarily in the testes of men, with smaller amounts produced in the adrenal glands. Testosterone plays a vital role in the development and maintenance of male sexual characteristics, including voice deepening, facial and body hair growth, and the maturation of male reproductive organs. This hormone has been linked to numerous medical conditions.

[0004] Currently, significant efforts are underway to develop therapies and treatments for ailments such as hypogonadism, polycystic ovarian syndrome (PCOS), various cancers, osteoporosis, and conditions related to virilization and masculinization. Additionally, hormone replacement therapies have gained widespread use. As a result, improved therapeutic modalities and precise diagnosis and continuous monitoring of hormone levels are needed in the formulation of effective therapeutic interventions.

[0005] While anti-testosterone antibodies have been reported in the art, there remains an unmet need for antibodies distinguished by their strong affinity and precise recognition of testosterone in the development of therapeutic agents and for high-sensitivity diagnostic tests for the precise quantification of hormone levels in biological specimens.SUMMARY OF THE INVENTION

[0006] In various embodiments, the present disclosure provides an anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0007] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, as defined by the IMGT numbering system. In some embodiments, the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 326-348 or SEQ ID NOs: 473-595.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 321, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 322, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 323, as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and an amino acid sequence selected from any one of SEQ ID NOs: 326-348 (LCDR3), as defined by the IMGT numbering system.

[0009] In various embodiments, the present disclosure provides an anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3comprises the amino acid sequence of SEQ ID NO: 470, as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 473-595 (LCDR3), as defined by the IMGT numbering system.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 350. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-319 or SEQ ID NOs: 351-462.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid of SEQ ID NO: 350, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0014] In various embodiments, the present disclosure provides an anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprisesan amino acid sequence of SEQ ID NO: 464, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G. In some embodiments, the antibody or antigenbinding fragment comprises an amino acid sequence of SEQ ID NO: 464, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, as defined by the IMGT numbering system. In some embodiments, the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 326-348 or SEQ ID NOs: 473-595.

[0016] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 464 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and an amino acid sequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 351-462 (LCDR3), as defined by the IMGT numbering system.

[0017] In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 466. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-319 or SEQ ID NOs: 351-462.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W,A, S, or E; and X6= E, H, T, C, Y, D, or G. In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0019] In some embodiments, the present disclosure provides an anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 465, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 465, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, as defined by the IMGT numbering system. In some embodiments, the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 326-348 or SEQ ID NOs: 473-595.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 465 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and an amino acidsequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 351-462 (LCDR3), as defined by the IMGT numbering system.

[0022] In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 467. In some embodiments, the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from any one of SEQ ID NO: 1-319 or SEQ ID NOs: 351-462. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0023] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319 or SEQ ID NOs: 351-462. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1-319.

[0024] In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 326 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 327 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 328 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 329 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 330 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 331 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 332 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 333 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 334 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising aminoacid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 335 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 336 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 337 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 338 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 339 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 340 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 341 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325(LCDR2), SEQ ID NO: 342 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 343 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 344 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 345 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 346 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 347 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 348 (LCDR3), as defined by the IMGT numbering system. In some embodiments, the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO:324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system.

[0025] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 12, 14, 16, 20, 21, 25, 29, 42, 54, 62, 69, 87, 106, 140, 195, 226, 233, 266, 270, 311, or 317.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 9. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 14. In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 25. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 29. In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprisingan amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 54. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 62. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 69. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 87. In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 106. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 140. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 195. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 226. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 270. In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 311. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO : 317. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 1.

[0027] In some embodiments, the present disclosure provides an antibody or antigen-binding fragment that competes for binding to testosterone with any one of the antibodies or antigenbinding fragments as disclosed herein.

[0028] In various embodiments, the present disclosure provides an anti-testosterone antibody or antigen-binding fragment that binds to the ketone group at the 3-position of testosterone and / or the hydroxyl group at the 17-position of testosterone.

[0029] In various embodiments, the present disclosure provides methods of treating a subject in need of inhibition of testosterone, comprising administering to the patient a therapeutically effective amount of an antibody or antigen-binding fragment as disclosed herein.In various embodiments, the present disclosure provides methods of detecting testosterone in a patient, comprising providing a biological sample from the patient and contacting the biological sample with an antibody or antigen-binding fragment as disclosed herein, wherein the antibody or antigen-binding fragment binds to testosterone and thereby detects the presence of testosterone in the sample. In some embodiments, the antibody or antigen-binding fragment comprises a detectable label. In some embodiments, the detection method comprises an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the detection method comprises a chemiluminescent immunoassay (CLIA). In some embodiments, the detection method comprises a radioimmunoassay (RIA). In some embodiments, the detection method comprises a co-immunoprecipitation assay (Co-IP). In some embodiments, the detection method comprises a lateral flow assay (LFA).BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 shows the structure of testosterone.

[0031] FIG. 2 shows an amino acid preference map for the ketone group at the 3-position of testosterone.

[0032] FIG. 3 shows an amino acid preference map for the hydroxyl group at the 17-position of testosterone.

[0033] FIG. 4 shows a flowchart of a sequence generation algorithm, prediction of antibody structure, and docking. Docking involves predicting how two molecules (e.g., testosterone and an antibody) interact and fit together in the pocket.

[0034] FIG. 5 shows a probability density plot for amino acids interacting with C3 of testosterone.

[0035] FIG. 6 shows binding of antibodies with the listed light chain sequences to testosterone-BSA (bovine serum albumin) conjugates in a lateral flow assay.

[0036] FIG. 7 shows a dendrogram, illustrating hierarchical clustering by amino acid mutation. The black arrow shows the threshold used for the Hamming distance.DETAILED DESCRIPTION

[0037] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to testosterone. Various exemplary embodiments are provided more fully with reference to the accompanying drawings, in which the various embodiments of the disclosure are shown. The disclosure may, however, be presented in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure is thorough and complete, and fully conveys the scope of the disclosure to those skilled in the art. In the drawings, the sizes of components may be exaggerated for clarity.

[0038] Generally, the various embodiments including the exemplary embodiments relate to the antibody or antigen-binding fragment for binding to testosterone.Definitions

[0039] Various terms relating to embodiments of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0040] As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

[0041] The terms “about” or “approximately” in the context of numerical values and ranges refer to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, such as having a desired amount of nucleic acids or polypeptides in a reaction mixture, as is apparent to the skilled person from the teachings contained herein. In some embodiments, “about” means plus or minus 10% of a numerical amount.

[0042] The term “antibody” is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least oneantigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain (HC) of an antibody is composed of a heavy chain variable domain (VH) and a heavy chain constant region (CH). The light chain (LC) is composed of a light chain variable domain (VL) and a light chain constant domain (CL). As used herein, the terms “domain” and “region” may be used interchangeably (e.g., the term “variable domain” may be used interchangeably with the term “variable region” and understood to refer to the same part of the antibody). For the purposes of this application, the mature heavy chain and light chain variable domains each comprise three complementarity determining regions (CDR1, CDR2, and CDR3) (also referred to as “hypervariable regions”) within four framework regions (FR1, FR2, FR3, and FR4) arranged from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 unless context indicates otherwise. CDRs may be identified according to the Kabat and / or IMGT numbering systems (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991); International ImMunoGeneTics Information System (IMGT®)). An “antibody” can be naturally occurring or man-made, such as monoclonal antibodies produced by conventional hybridoma technology. The term “antibody” includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antigen-binding fragments such as Fab, Fab', F(ab')2, Fv, and single chain antibodies. An antibody can be any one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., isotypes IgGl, IgG2, IgG3, IgG4). An antibody of any of the aforementioned class or subclass can also comprise one of two functionally similar classes of light chains: IgK (also referred to herein as “Ig kappa” or “kappa”) and Ig / . (also referred to herein as “Ig lambda” or “lambda”). The term antibody encompasses human antibodies, chimeric antibodies, humanized antibodies, and any modified immunoglobulin molecule containing an antigen recognition site, so long as it demonstrates the desired biological activity.

[0043] The term “human antibody,” as used herein, refers to an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.

[0044] The term “antigen-binding fragment” or “antigen-binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., testosterone). Antigen-binding fragments preferably also retain the ability to internalize into an antigen-expressing cell. In some embodiments, antigen-binding fragments also retain immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” or “antigenbinding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and Cm domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and Cm domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment, which comprises a single variable domain, e.g., a VH domain (see, e.g., Ward et al. (1989) Nature 341:544-6; and Winter et al., WO 90 / 05144); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). See, e.g., Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment” or “antigenbinding portion” of an antibody, and are known in the art as an exemplary type of binding fragment that can internalize into cells upon binding. See, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J. Nucl. Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402. In certain embodiments, scFv molecules may be incorporated into a fusion protein. Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites. See, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 8; and Poljak et al. (1994) Structure 2: 1121-3. Antigen-binding fragments are obtained using conventional techniques known to those of skill in the art.

[0045] The term “anti-testosterone antibody” or “antibody that specifically binds testosterone” refers to any form of antibody or fragment thereof that specifically binds testosterone and encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and biologically functional antigen-binding fragments so long as they specifically bind testosterone. As used herein, the terms “specific,” “specificallybinds,” and “binds specifically” refer to the selective binding of the antibody to the target antigen or epitope over alternative antigens or epitopes. Antibodies can be tested for specificity of binding by comparing binding to an appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen with at least 5 times, or preferably 7 or 10 times, e.g., with a KD of at most 10 nM, more affinity than to an irrelevant antigen or antigen mixture, then it is considered to be specific, e.g., as measured by surface plasmon resonance. In one embodiment, a specific antibody is one that binds the testosterone antigen but does not bind (or exhibits minimal binding) to other antigens.

[0046] Generally, variants of antibodies disclosed herein retain testosterone binding. Such variants typically have amino acid homology, similarity, or identity of at least 80%, and more typically homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and almost 100%, or 100%, to at least the six CD Rs of a given antibody, or to the heavy and / or light chain variable domains.

[0047] For amino acid sequences, sequence identity and / or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al. (1984) Nucl. Acid Res. 12:387-95, preferably using the default settings, or by inspection.

[0048] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J. Mol. Evol. 35 :351-60; the method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0049] Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al. (1990) J. Mol. Biol. 215:403-10; Altschul et al. (1997) Nucleic Acids Res. 25:3389-402; and Karin et al. (1993) Proc. Natl. Acad. Sci. USA 90:5873-87. A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

[0050] An additional useful algorithm is gapped BLAST as reported by Altschul et al. (1993) Nucl. Acids Res. 25:3389-402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to about 22 bits.

[0051] The term “epitope” refers to the portion of an antigen capable of being recognized and specifically bound by an antibody. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the polypeptide. The epitope bound by an antibody may be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variations of the antigen, or monitoring solvent accessibility of different parts of the antibody and the antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligo-peptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (See, e.g., Gershoni et al. (2007) 21: 145-56; and Hager-Braun and Tomer (2005) Expert Rev. Proteomics 2:745-56).

[0052] Competitive binding and epitope binning can also be used to determine antibodies sharing identical or overlapping epitopes. Competitive binding can be evaluated using across-blocking assay, such as the assay described in “Antibodies, A Laboratory Manual,” Cold Spring Harbor Laboratory, Harlow and Lane (1st edition 1988, 2nd edition 2014). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces binding of a reference antibody or binding protein to a target antigen such as testosterone (e.g., a binding protein comprising CDRs and / or variable domains selected from those identified in Tables 2, 4, and 6), by at least about 50% in the cross-blocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between), and / or vice versa. In some embodiments, competitive binding can be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance where antibodies or binding proteins bind at nearby epitopes. See, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66). In some embodiments, competitive binding can be used to sort groups of binding proteins that share similar epitopes, e.g., those that compete for binding can be “binned” as a group of binding proteins that have overlapping or nearby epitopes, while those that do not compete are placed in a separate group of binding proteins that do not have overlapping or nearby epitopes.

[0053] The term “KD” refers to the equilibrium dissociation constant of a particular antibodyantigen interaction. KD is calculated by ka / kd. The rate can be determined using standard assays, such as surface plasmon resonance.

[0054] The term “ka” refers to the on-rate constant for association of an antibody to the antigen to form the antibody / antigen complex. The rate can be determined using standard assays, such as surface plasmon resonance.

[0055] The term “kd” refers to the off-rate constant for dissociation of an antibody from the antibody / antigen complex. The rate can be determined using standard assays, such as surface plasmon resonance.

[0056] The use of “or” will mean “and / or” unless the specific context of its use dictates otherwise.Anti-Testosterone Antibodies or Antigen-Binding Fragments

[0057] The present disclosure provides antibodies or antigen-binding fragments that specifically bind to testosterone and may be used alone or in combination, e.g., formulated as antibody compositions, e.g., for therapeutic use, or as diagnostics for use in detectinghormones. Conjugates and combinations with the disclosed antibodies are also contemplated. For example, an antibody may be conjugated to a detectable label or to an additional therapeutic agent.

[0058] In some embodiments, the antibody of the present invention may include any type of antibodies. Examples of the antibody may include, but not limited to, a monoclonal antibody, polyclonal antibody, mouse antibody, chimeric antibody, single chain, multi chain, humanized antibody, murine, fully human, binding fragments, and the like. Embodiments of the present disclosure are intended to include or otherwise cover any type of the antibody including known, related art, and / or later developed antibody, without deviating from the scope of the present disclosure. In some embodiments, the monoclonal antibody may include, but not limited to, naked monoclonal antibodies, conjugated monoclonal antibodies, bispecific monoclonal antibodies, and the like.

[0059] In some embodiments, the antibody or antigen-binding fragment may be of recombinant sources and / or animal sources such as mice, rabbit, sharks, chickens, hamster, goats, horses, rat, and the like. Embodiments of the present disclosure are intended to include or otherwise cover any type of the source of the antibody including known, related art, and / or later developed sources, without deviating from the scope of the present disclosure.

[0060] In some embodiments, the backbone of the antibody or antigen-binding fragment may be selected from the group, but are not limited to, a mouse, rat, rabbit, horse, hamster, guinea pig, human, IgA, IgB, IgG, IgM, IgD, IgE and the like. Embodiments of the present disclosure are intended to include or otherwise cover any type of the backbone including known, related art, and / or later developed backbones, without deviating from the scope of the present disclosure.

[0061] In some embodiments, the antibody or antigen-binding fragment may specifically bind to testosterone. Testosterone is a 4-ringed steroid molecule from the androstane class of steroids containing a ketone at the 3’ positions and a hydroxyl at the 17-position (FIG. 1). Atoms in testosterone, as referenced herein, are numbered as shown below:

[0062] In some embodiments, the light chain of the antibody or antigen-binding fragment may recognize and bind to the group at the 17-position of the testosterone. In some embodiments, the antibody or the antigen-binding fragments of the present disclosure may include a light chain sequence set forth in SEQ ID NO: 1.

[0063] Antibody mentioned here refers to a canonical structure comprising a heavy chain region and a light chain region. The heavy chain comprises of a variable region and a constant region while the light chain consists of a variable region. The steroid molecule (in this case testosterone) binds to the hypervariable loop regions in the CDR3 of light chain and heavy chain. The sequences identified are light chain CDR3 (LCDR3) sequences which can interact with C3 / C17 of the testosterone molecule which is the feature that differentiates testosterone from other steroid molecules.

[0064] In various embodiments, an antibody or antigen -binding fragment as disclosed herein may comprise an LCDR3 as disclosed herein, e.g., in any of Tables 1 or 4, in combination with any other suitable CDR, framework, and / or constant domain sequences. In some embodiments, an antibody that specifically binds testosterone comprises a light chain having an LCDR3 comprising the amino acid sequence of SEQ ID NO: 349. In some embodiments, an antibody that specifically binds testosterone comprises a light chain having an LCDR3 comprising the amino acid sequence of SEQ ID NO: 462. In some embodiments, an antibody that specifically binds testosterone comprises a light chain having an LCDR3 comprising an amino acid sequence selected from SEQ ID NOs: 473-521. In some embodiments, an antibody that specifically binds testosterone comprises a light chain having an LCDR3 comprising the amino acid sequence of SEQ ID NO: 463. In some embodiments, an antibody that specifically binds testosterone comprises a light chain having an LCDR3 comprising an amino acid sequence selected from Table 4. In some embodiments, an antibody that specifically binds testosteronecomprises a light chain having an LCDR3 comprising amino acid sequence selected from SEQ ID NOs: 326-348 or SEQ ID NOs: 473-595.

[0065] In various embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs (HCDRs) and three light chain CDRs (LCDRs), as defined by the IMGT numbering system.

[0066] In various embodiments, an antibody or antigen -binding fragment as disclosed herein may comprise a light chain as disclosed herein, e.g., in any of Tables 5-7, in combination with any other suitable heavy chain sequence. In some embodiments, an antibody that specifically binds testosterone comprises a light chain comprising an amino acid sequence selected from any of SEQ ID NOs: 1-319 or SEQ ID NOs: 351-462. In some embodiments, an antibody that specifically binds testosterone comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or SEQ ID NO: 463. In some embodiments, an antibody that specifically binds testosterone comprises a light chain comprising an amino acid sequence selected from any of SEQ ID NOs: 1-319 or SEQ ID NOs: 351-462 in combination with a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or SEQ ID NO: 463. In some embodiments, an antibody that specifically binds testosterone comprises a light chain comprising an amino acid sequence selected from the sequences in Table 6 in combination with a heavy chain comprising the amino acid sequence of SEQ ID NO: 320 or SEQ ID NO: 463. In some embodiments, an antibody that specifically binds testosterone comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1-319. In some embodiments, an antibody that specifically binds testosterone comprising a heavy chain having an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of any of SEQ ID NOs: 351-462.

[0067] The light chain sequences included in Table 6 (SEQ ID NOs: 351-399) have an isoelectric point (pl) of 5.5-8.5 (e.g., a pH suitable for diagnostic assays and / or in vivo use). The light chain sequences included in Table 7 (SEQ ID NOs: 400-462) have a pl of 5.5-9.5.

[0068] Light chain (SEQ ID NO: 1):DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCFQFSHDPTTFGGGTKLEIK

[0069] Heavy chain (SEO ID NO: 320):QVQLQESGGGLVNLGGSMTLSCVASGFTFNTYYMSWVRQTPEKTLELVAAINSDGE PIYYPDTLKGRVTISRDNAKKTLYLQMSSLNFEDTALYYCARLNYAVYGMDYWGQ GTTVTVSS

[0070] Heavy chain (SEO ID NO: 463):EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYALSWVRQTADKRLEWVASIVSGGN TYYSGSVKGRFTISRDIARNILYLQMSSLRSEDTAMYYCAREYYGYVGLAYWGQGT LVTVSAA

[0071] Amino acid sequences of exemplary antibodies of the present disclosure are set forth in Tables 1-7. The monoclonal antibody IMGT CDR and full-chain consensus sequences reflect the alignment of the light chain CDR3 (LCDR3) sequences represented by SEQ ID NOs: 326-348 and SEQ NOs: 473-595 and the light chain sequences represented by SEQ ID NOs: 1-319 and SEQ ID NOs 351-462, respectively. Residues that differ between clones are represented by “X” in SEQ ID NOs: 349, 350, and 464-467. In some embodiments, an antitestosterone antibody or antigen-binding fragment as described herein may be defined by the consensus CDR sequences of Table 1 in combination with the CDR sequences of Table 3, e.g., by selecting an LCDR3 sequence of Table 1 and a HCDR1, HCDR2, HCDR3, LCDR1, and LCDR2 of T able 3 to describe an antibody by its three heavy chain and three light chain CDR sequences. In some embodiments, an anti-testosterone antibody or antigen-binding fragment as described herein comprises a consensus light chain sequence selected from Table 2 in combination with a heavy chain having an amino acid sequence of SEQ ID NO: 320 or SEQ ID NO: 463.Table 1. Amino acid sequences in consensus antibody CDRsX1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D; X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; X6= H, Y, T, C, E, G, D, L, F, or I; X7is F, T, D, E, G, K, L, V Y, Q, orN; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or YTable 2. Amino acid sequences of consensus full-length antibody Ig chainsX1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D; X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; X6= H, Y, T, C, E, G, D, L, F, or I; X7is F, T, D, E, G, K, L, V Y, Q, orN; X8= F, T, D, E, G, K, L, V, Y, Q, orN; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or YTable 3. Amino acid sequences of antibody CDRsTable 4. Amino acid sequences of antibody LCDR3

[0072] SEQ ID NOs: 2-319 comprise light chain sequence of SEQ ID NO: 1 with substitutions at positions 96, 99, and 101. Table 5 indicates the residues at positions 96-112 for each full- length sequence with the bolded residues indicating a substitution relative to SEQ ID NO: 1.Table 5. Amino acid sequences of full-length antibody Ig chainsBE= binding energyTable 6. Amino acid sequences of full-length antibody Ig chainsTable 7. Amino acid sequences of full-length antibody Ig chains

[0073] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0074] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0075] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0076] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 464, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0077] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 464, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

[0078] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 465, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

[0079] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 465, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0080] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 321, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 322, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 323, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 349, as defined by the IMGT numbering system, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0081] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 349, whereinX1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0082] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0083] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 349, as defined by the IMGT numbering system, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0084] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 349, as defined by the IMGT numbering system, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0085] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0086] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO:469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 349, as defined by the IMGT numbering system, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0087] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 349, as defined by the IMGT numbering system, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0088] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0089] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 464, as defined by the IMGT numbering system, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0090] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 464, as defined by the IMGT numbering system, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0091] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO:464 (LCDR3), as defined by the IMGT numbering system, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0092] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 464, as defined by the IMGT numbering system, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

[0093] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 464, as defined by the IMGT numbering system, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

[0094] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 464 (LCDR3), as defined by the IMGT numbering system, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

[0095] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 465, as defined by the IMGT numbering system, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

[0096] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 465, asdefined by the IMGT numbering system, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

[0097] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 465 (LCDR3), as defined by the IMGT numbering system, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

[0098] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 465, as defined by the IMGT numbering system, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0099] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463, and the light chain comprising an LCDR3 comprising the amino acid sequence of SEQ ID NO: 465, as defined by the IMGT numbering system, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V Y.

[0100] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 465 (LCDR3), as defined by the IMGT numbering system, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0101] In some embodiments, the anti-testosterone testosterone antibody or antigen-binding fragment comprises wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2),and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and an amino acid sequence selected from SEQ ID NOs: 326-348 (LCDR3), as defined by the IMGT numbering system.

[0102] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NOs: 326-348 or SEQ ID NOs: 473-595 (LCDR3), as defined by the IMGT numbering system.

[0103] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a light chain comprising an amino acid of SEQ ID NO: 350, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0104] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0105] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0106] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

[0107] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0108] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

[0109] In some embodiments, the anti-testosterone antibody or antigen-binding fragment a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is T, F, D, E,G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0110] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid of SEQ ID NO: 350, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

[0111] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

[0112] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, C, D, E, F, G,H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

[0113] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

[0114] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

[0115] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

[0116] In some embodiments, the anti-testosterone antibody or antigen -binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is T, F, D, E, G, K,L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

[0117] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319 or SEQ ID NOs: 351-462.

[0118] In some embodiments, the anti-testosterone antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1-319.

[0119] In some embodiments, the antibody or antigen -binding fragments may be provided for managing a disease or disorder in a subject by way of therapy. Examples of testosterone-related diseases or disorders include, but are not limited to, hypogonadism, PCOS, various cancers, osteoporosis, and conditions related to virilization and masculinization. The antibodies could be used to treat PCOS, the major symptoms of which are due to high amounts of testosterone. See, e.g., Simons et al. (2021) Endocrinol. Diab. Metab. 4:e00267; Nelson et al. (2001) J. Clin. Endocrinol. Metab. 86(12):5925-33 ; and Grassi et al. (2021) J. Clin. Med. 10(l):119. Humanised versions for these antibodies could be used to bind to excessive free testosterone in the body and drive it out of the body. Similarly, these antibodies could be used to treat androgen-dependent alopecia which is also caused due to excess testosterone. See, e.g., Price (1975) Arch. Dermatol. 11(11): 1496-1502; Hoffmann et al. (2002) Clin. Exp. Dermtaol. 27(5):373-82; and Ralph (2002) Exp. Gerontol. 37(8-9):981-90. Such antibodies can also be used to slow down the growth of testosterone-dependent prostate cancer cells by reducing excess amounts of testosterone in males. See, e.g., Michaud et al. (2015) Then Adv. Urol. 7(6):378-87; Morgentaler et al. (2009) Eur. Urol. 55(2):310-20; Yassin et al. (2019). Aging Male 22(4):219-227; and Klap et al. (2015) J. Urol. 193(2):403-13. Furthermore, these antibodies could be used to reduce testosterone and promote estrogen production in transgender women.

[0120] In some embodiments, a method for treating a subject may be provided. The method may include administering an antibody or antigen-binding fragment thereof as disclosed herein to a subject in need thereof. The antibody may be administered in a suitable pharmaceutical composition and by a suitable route of administration known in the art.

[0121] In some embodiments, a pharmaceutical composition may be provided. The pharmaceutical composition may include an antibody or antigen-binding fragment thereof as disclosed herein for administration to a subject for managing treatment of a disease or disorder and a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise one or more additional active agents for treating one of the indications mentioned above, or may be administered in conjunction with such an additional active agent.Diagnostic Uses

[0122] In some embodiments, the anti-testosterone antibodies or antigen-binding fragments of the present disclosure may be provided for detection or diagnosis of testosterone- related disorders or diseases.

[0123] In some embodiments, the antibody or antigen-binding fragment may bind to a target molecule, e.g., testosterone, present in a sample. In some embodiments of the present disclosure, the sample may be a bodily fluid. Examples of the sample may include, but are not limited to, aqueous humor, vitreous humor, bile, blood, cerebrospinal fluid, chyle, endolymph, perilymph, female ejaculate, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sputum, synovial fluid, vaginal secretion, semen, blood, serum, plasma, and the like. Embodiments of the present disclosure are intended to include or otherwise cover any type of the sample including known, related art, and / or later developed sample without deviating from the scope of the present disclosure.

[0124] In some embodiments, a method for detection of testosterone in a sample may be provided. The method may include an antibody or antigen-binding fragment thereof as disclosed herein such that the antibody or antigen-binding fragment may specifically recognise and bind to testosterone present in the sample. This can be determined by standard methods known in the art, such as ELISA assay, radioimmunoassays, lateral flow assays, or other assays with a fluorescently labelled antibody.

[0125] In some embodiments, a method for selection of therapy to a subject in need of therapy for an abnormal level of testosterone is provided. The method may include determining or monitoring the levels or concentrations of a testosterone in sample obtained from a diseased patient by detecting and quantifying the amount of an antibody or antigen-binding fragment thereof as disclosed herein bound to the target molecule of interest present in the sample.Examples of testosterone-related diseases or disorders include, but are not limited to, hypogonadism, PCOS, various cancers, osteoporosis, and conditions related to virilization and masculinization. In some embodiments, a therapy is provided when an abnormal level of testosterone is detected in the sample.

[0126] In some embodiments, an assay for quantification of an analyte in a sample may be provided, such that the analyte may be testosterone. The assay may include an antibody or antigen-binding fragment thereof as disclosed herein, such that the antibody or antigen -binding fragment may specifically recognise and bind to testosterone present in the sample. Examples of assay may include, but not limited to, a sandwich assay, multiplexed and individual competitive assay, non- competitive assay, lateral flow assay, membrane-based assay, immunoassay, multiplexed sandwich assay, individual sandwich assay, and the like. Aspects of the present disclosure are intended to include or otherwise cover any type of assay including known, related art, and / or later developed assay, without deviating from the scope of the present disclosure.

[0127] In some embodiments, a method for determining one or more health conditions of a female subject may be provided. The method may include determining concentration, levels, or rate of change in concentration levels of a target molecule, more particularly testosterone in a sample based on the binding of an antibody or antigen-binding fragment thereof as disclosed herein to the target molecules present in the sample.

[0128] In some embodiments, an antibody or antigen-binding fragment thereof as disclosed herein may be impregnated on immunochromatographic test strips such that the antibody or antigen-binding fragment may specifically bind to testosterone present in the sample. In some embodiments, the test strip may include one or more lateral flow assays. In some embodiments, the one or more lateral assays may include both a competitive and sandwich assay format. In some embodiments, the lateral flow assay may include a single assay for measuring one or more analytes in a competitive and sandwich assay format.

[0129] The foregoing discussion of the present disclosure has been presented for purposes of illustration and description. It is not intended to limit the present disclosure to the form or forms disclosed herein. In the foregoing Detailed Description, for example, various features of the present disclosure are grouped together in one or more embodiments, configurations, or embodiments for the purpose of streamlining the disclosure. The features of the embodiments,configurations, or embodiments may be combined in alternate embodiments, configurations, or embodiments other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention, the present disclosure requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive embodiments lie in less than all features of a single foregoing disclosed aspect, configuration, or aspect. Thus, the following claims are hereby incorporated into this Detailed Description, with each claim standing on its own as a separate aspect of the present disclosure.

[0130] Moreover, though the description of the present disclosure has included a description of one or more embodiments, configurations, or embodiments and certain variations and modifications, other variations, combinations, and modifications are within the scope of the present disclosure, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights that include alternative embodiments, configurations, or embodiments to the extent permitted, including alternate, interchangeable, and / or equivalent structures, functions, ranges, or steps to those claimed, whether or not such alternate, interchangeable and / or equivalent structures, functions, ranges, or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.Enumberated Embodiments1. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.2. The anti-testosterone antibody or antigen-binding fragment of embodiment 1, wherein the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, as defined by the IMGT numbering system,3. The anti-testosterone antibody or antigen-binding fragment of embodiment 1 or embodiment 2, wherein LCDR3 comprises an amino acid sequence of SEQ ID NO: 349.4. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 4, wherein the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 326- 348.5. The anti-testo sterone antibody or antigen-binding fragment of any one of embodiments 1 to 4, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system.6. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 5, wherein the antibody or antigen -binding fragment comprises:(i) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:326 (LCDR3), as defined by the IMGT numbering system;(ii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:327 (LCDR3), as defined by the IMGT numbering system;(iii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:328 (LCDR3), as defined by the IMGT numbering system;(iv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:329 (LCDR3), as defined by the IMGT numbering system;(v) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:330 (LCDR3), as defined by the IMGT numbering system;(vi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:331 (LCDR3), as defined by the IMGT numbering system;(vii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:332 (LCDR3), as defined by the IMGT numbering system;(viii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:333 (LCDR3), as defined by the IMGT numbering system;(ix) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:334 (LCDR3), as defined by the IMGT numbering system;(x) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:335 (LCDR3), as defined by the IMGT numbering system;(xi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:336 (LCDR3), as defined by the IMGT numbering system;(xii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprisingamino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:337 (LCDR3), as defined by the IMGT numbering system;(xiii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:338 (LCDR3), as defined by the IMGT numbering system;(xiv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:339 (LCDR3), as defined by the IMGT numbering system;(xv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:340 (LCDR3), as defined by the IMGT numbering system;(xvi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:341 (LCDR3), as defined by the IMGT numbering system;(xvii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:342 (LCDR3), as defined by the IMGT numbering system;(xviii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:343 (LCDR3), as defined by the IMGT numbering system;(xix) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:344 (LCDR3), as defined by the IMGT numbering system;(xx) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:345 (LCDR3), as defined by the IMGT numbering system;(xxi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:346 (LCDR3), as defined by the IMGT numbering system;(xxii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:347 (LCDR3), as defined by the IMGT numbering system;(xxiii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:348 (LCDR3), as defined by the IMGT numbering system;(xxiv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:349 (LCDR3), as defined by the IMGT numbering system.7. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 6, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 350.8. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 7, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319.9. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 8, wherein the antibody or antigen-binding fragment comprises a heavy chain comprisingan amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 350, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.10. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 9, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319.11. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 10, wherein the antibody or antigen -binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 12, 14, 16, 20, 21, 25, 29, 42, 54, 62, 69, 87, 106, 140, 195, 226, 233, 266, 270, 311, or 317.12. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 1 to 11, wherein the antibody or antigen-binding fragment comprises:(i) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 5;(ii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 9;(Hi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 12;(iv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 14;(v) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 16;(vi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 20;(vii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 21;(viii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 25;(ix) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 29;(x) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 42;(xi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 54;(xii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 62;(xiii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 69;(xiv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 87;(xv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 106;(xvi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 140;(xvii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 195;(xviii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 226;(xix) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 270;(xx) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 311;(xxi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 317; or(xxii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 1.13. An antibody or antigen-binding fragment that competes for binding to testosterone with the antibody or antigen-binding fragment of any one of embodiments 1 to 12.14. An anti-testosterone antibody or antigen-binding fragment that binds to the ketone group at the 3-position of testosterone and / or the hydroxyl group at the 17-position of testosterone.15. The anti-testosterone antibody or antigen-binding fragment of embodiment 14, wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.16. The anti-testosterone antibody or antigen-binding fragment of embodiment 14 or embodiment 15, wherein the antibody or antigen -binding fragment comprises three HCDRs and three LCDRs, as defined by the IMGT numbering system,17. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 16, wherein LCDR3 comprises an amino acid sequence of SEQ ID NO: 349.18. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 17, wherein the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, as defined by the IMGT numbering system, and wherein the LCDR3 comprises an amino acid sequence selected from SEQ ID NO: 326-348.19. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 18, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system.20. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments14 to 19, wherein the antibody or antigen -binding fragment comprises:(i) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:326 (LCDR3), as defined by the IMGT numbering system;(ii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:327 (LCDR3), as defined by the IMGT numbering system;(iii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:328 (LCDR3), as defined by the IMGT numbering system;(iv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:329 (LCDR3), as defined by the IMGT numbering system;(v) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:330 (LCDR3), as defined by the IMGT numbering system;(vi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:331 (LCDR3), as defined by the IMGT numbering system;(vii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:332 (LCDR3), as defined by the IMGT numbering system;(viii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:333 (LCDR3), as defined by the IMGT numbering system;(ix) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:334 (LCDR3), as defined by the IMGT numbering system;(x) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:335 (LCDR3), as defined by the IMGT numbering system;(xi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:336 (LCDR3), as defined by the IMGT numbering system;(xii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:337 (LCDR3), as defined by the IMGT numbering system;(xiii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:338 (LCDR3), as defined by the IMGT numbering system;(xiv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:339 (LCDR3), as defined by the IMGT numbering system;(xv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprisingamino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:340 (LCDR3), as defined by the IMGT numbering system;(xvi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:341 (LCDR3), as defined by the IMGT numbering system;(xvii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:342 (LCDR3), as defined by the IMGT numbering system;(xviii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:343 (LCDR3), as defined by the IMGT numbering system;(xix) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:344 (LCDR3), as defined by the IMGT numbering system;(xx) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:345 (LCDR3), as defined by the IMGT numbering system;(xxi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:346 (LCDR3), as defined by the IMGT numbering system;(xxii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:347 (LCDR3), as defined by the IMGT numbering system;(xxiii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:348 (LCDR3), as defined by the IMGT numbering system;(xxiv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:349 (LCDR3), as defined by the IMGT numbering system.21. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 20, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 350.22. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 21, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319.23. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 22, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 350.24. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 23, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319.25. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments 14 to 24, wherein the antibody or antigen -binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an aminoacid sequence selected from SEQ ID NO: 1, 5, 9, 12, 14, 16, 20, 21, 25, 29, 42, 54, 62, 69, 87, 106, 140, 195, 226, 233, 266, 270, 311, or 317.26. The anti-testosterone antibody or antigen-binding fragment of any one of embodiments14 to 25, wherein the antibody or antigen-binding fragment comprises:(i) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 5;(ii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 9;(Hi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 12;(iv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 14;(v) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 16;(vi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 20;(vii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 21;(viii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 25;(ix) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 29;(x) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 42;(xi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 54;(xii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 62;(xiii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 69;(xiv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 87;(xv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 106;(xvi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 140;(xvii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 195;(xviii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 226;(xix) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 270;(xx) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 311;(xxi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 317; or(xxii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 1.27. A method of treating a subject in need of inhibition of testosterone, comprising administering to the patient a therapeutically effective amount of the antibody or antigenbinding fragment of any one of embodiments 1 to 26.28. A method of detecting testosterone in a patient, comprising providing a biological sample from the patient and contacting the biological sample with the antibody or antigenbinding fragment of any one of embodiments 1 to 26, wherein the antibody or antigen-binding fragment binds to testosterone and thereby detects the presence of testosterone in the sample.29. The method of embodiment 28, wherein the antibody or antigen-binding fragment comprises a detectable label.30. The method of embodiment 29, wherein the detection method comprises an enzyme- linked immunosorbent assay (ELISA).31. The method of embodiment 29, wherein the detection method comprises a chemiluminescent immunoassay (CLIA).32. The method of embodiment 29, wherein the detection method comprises a radioimmunoassay (RIA).33. The method of embodiment 29, wherein the detection method comprises a coimmunoprecipitation assay (Co-IP).EXAMPLES

[0131] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to methods, and experimental conditions described, as such methods and conditions may vary.Example 1: Creation of Amino-Acid Preference Map Algorithm

[0132] To minimize the number of amino acids used for mutations, an amino acid preference map was created that identifies amino acids with a high affinity towards specific atoms of sterol molecules, such as testosterone. This example outlines the process used to generate a mutational space aimed at reducing the large combinatorial space of amino acids. The goal is to identify amino acids with high affinity towards sterol molecules. The method calculates the probability of occurrence of all amino acids in proximity to each atom of a particular ligand.The results are displayed as a bar graph of amino acids vs their probabilities of occurrence, referred to as the "amino acid preference map.”Selection of Target Groups

[0133] We developed amino acid preference charts for the ketone group at the 3-position and the hydroxyl group at the 17-position of testosterone. The ketone and hydroxyl groups were chosen because of their oxygen atoms, which, due to their electronegativity and ability to form hydrogen bonds, contribute significantly to binding affinity compared to carbon atoms.Identification of Substructures

[0134] Substructures of testosterone refer to the distinct chemical components and functional groups that make up the overall molecular structure of testosterone. Testosterone is a steroid hormone derived from cholesterol, and its structure can be divided into several key substructures:• Steroid Backbone: Testosterone has a characteristic four-ring steroid backbone structure, consisting of three six-membered cyclohexane rings (designated as rings A, B, and C) and one five-membered cyclopentane ring (designated as ring D).• Hydroxyl Group: There is a hydroxyl (OH) group attached to the third carbon atom in the A-ring. This hydroxyl group plays a crucial role in the biological activity of testosterone.• Keto Group: A carbonyl (C=O) group is attached to the seventeenth carbon atom in the D-ring. This keto group is another essential functional group contributing to the hormone's properties.• Methyl Groups: Two methyl groups (CH3) are present in the testosterone molecule. One is attached to the tenth carbon atom (CIO) and the other to the thirteenth carbon atom (C13).• Double Bonds: Testosterone has a double bond between the fourth and fifth carbon atoms (C4 and C5) in the A-ring.• Alkyl Side Chain: A side chain, specifically a hydrocarbon chain, is attached to the seventeenth carbon atom, which is typical of steroids.

[0135] Structures of testosterone with a ketone group at the 3-position of the sterol ring and a hydroxyl group at the 17-position of the sterol ring were identified, and their Chemical IDs were determined.Complex Identification and Interaction Extraction

[0136] A search was then conducted to find complexes in nature associated with these Chemical IDs. Using the complex’s Protein Data Bank (PDB) files, the interactions between ligand atoms and amino acids, including hydrogen bonds and non-bonded contacts, were determined. Amino acids interacting with the 3 -position oxygen atom of the ketone group were then extracted from all PDB entries. The occurrences of different amino acids were counted separately for hydrogen bonds and non-bonded contacts.Amino Acid Selection and Permutation

[0137] The occurrence numbers were converted into a probability density plot for amino acid occurrences at the 3-position oxygen atom and the hydroxyl group at the 17-position of testosterone. All amino acids with probability scores above a certain threshold were selected and permuted on known flexible residue locations to generate N number of mutated sequences.

[0138] This method effectively narrowed down the amino acids used for mutations by focusing on those with the highest probability of interaction with key functional groups of the ligand. The generated amino acid preference maps provide a visual and quantitative basis for selecting amino acids for protein engineering and mutagenesis studies. Focusing on substructures like the ketone group at the 3-position (FIG. 2) and the hydroxyl group at the 17- position (FIG. 3) and leveraging the specific binding properties of these functional groups improves the accuracy and efficiency of this mutational analysis.Example 2: Schema of Identification of Best Binding Sequences

[0139] Using the Amino-Acid Preference Maps from Example 1, the residues that interact with testosterone were identified in the consensus sequence by initial blind docking as shown in Table 8.Table 8. Residues that Interact with Testosterone

[0140] The amino acids in the estimated active sites of wild-type light chain sequence were mutated to new amino acids based on the Amino-Acid Preference Map from Example 1. The wild-type heavy chain was retained.Protein Folding Algorithms

[0141] Protein folding algorithms are computational methods used to predict the three- dimensional structure of a protein from its amino acid sequence. These structures are crucial for understanding the protein's function and interactions. Two prominent protein folding algorithms mentioned are ImmuneBuilder and AlphaFold. ImmuneBuilder is specifically designed for modeling antibody structures. AlphaFold, developed by DeepMind, is a general protein structure prediction algorithm known for its high accuracy.

[0142] The Protein Data Bank (PDB) file format is a standard format for representing three- dimensional structures of molecules. By passing the heavy and light chain sequences through protein folding algorithms, a PDB file can be generated that contains the 3D coordinates of the atoms in the folded protein.

[0143] The algorithms used are described below.1. Pass this folded protein(pdb) & testosterone molecule(mol2 / sdf) through the docking pipeline. a. Input Preparation i. Prepare Protein (Receptor) File:1. Input: Protein Data Bank (PDB) file.2. Process: o Clean the protein structure by removing water molecules and other non-essential components.o Add polar hydrogen atoms. o Assign appropriate partial charges (e.g., using the Gasteiger method).3. Output: Protein structure in PDBQT format.4. Tools: AutoDockTools (ADT) ii. Prepare Ligand File:1. Input: M0L2 file of the ligand.2. Process: o Add polar hydrogen atoms. o Assign appropriate partial charges. o Set rotatable bonds.3. Output: Ligand structure in PDBQT format.4. Tools: AutoDockTools (ADT). b. Grid Parameter File (GPF) Preparation i. Define Grid Box:1. Input: Prepared PDBQT file of the protein.2. Process: o Define the docking box dimensions and center around the active site or the whole protein. o Set the grid spacing.3. Output: Grid parameter file (GPF).4. Tools: AutoGrid, AutoDockTools (ADT) c. Execution of Docking i. Run AutoGrid:1. Input: Grid parameter file (GPF) and protein PDBQT file.2. Process: o Generate grid maps.3. Output: Grid map files.4. Tools: AutoGrid. n. Run AutoDock:1. Input: Docking parameter file (GLG), grid map files, and ligand PDBQT file.2. Process: o Execute the docking simulation. o Output: Docking log file (DLG) containing docking results. o Tools: AutoDock. d. Post-Processing and Analysis i. Analyze Docking Results:1. Input: Docking log file (DLG).2. Process: o Extract binding poses and binding affinities. o Rank binding poses based on binding energy. o Output: Ranked list of binding poses and corresponding binding affinities. o Tools: AutoDockTools (ADT), custom scripts for further analysis. ii. Visualize Docking Results:1. Input: Docked conformations (from DLG) and protein PDB file.2. Process: o Visualize docking results to confirm binding mode and interactions. o Output: Visual representation of docked poses. o Tools: PyMOL, Discovery, AutoDockTools (ADT).2. Iterate over all the sequences created using Amino-Acids created using Amino-Acid Preference Map created in Step 1.

[0144] After going through all the possible mutations, analysing the binding poses, and verifying the key interactions (hydrogen bonds and Van der Waals interactions), the top 5 mutants with the lowest binding affinity were selected.Example 3: Mutation Experiment Workflow

[0145] The systematic approach for conducting mutation experiments on a protein model aims to enhance its binding affinity to testosterone is depicted in FIG. 4. The process is organized in sequential steps and continues through iterative mutation cycles. The goal was to identify and optimize critical residue interactions within the protein structure.Example 4: Identification of Representative Sequences for Experimental Validation

[0146] A set of l ' l representative sequences were chosen from the identified antibody sequences by focusing on the LCDR3 region, specifically at positions 96, 99, and 101. Position 96 can be occupied by a non-polar, basic, or aromatic amino acid; position 99 by an acidic, basic, or non-polar amino acid; and position 101 by an aromatic, basic, or non-polar amino acid. Despite the variability within these amino acid groups, one representative amino acid was selected from each category for each position. This systematic selection process ensured that all possible combinations of these representative amino acids were included, resulting in a comprehensive set of 27 sequences. This approach maintains the diversity of the original dataset while simplifying the analysis and application of these sequences in further research and development.

[0147] The representative sequences chosen were SEQ ID NO: 5, SEQ ID NO: 14, SEQ ID NO: 54, SEQ ID NO: 87, SEQ ID NO: 106, SEQ ID NO: 140, SEQ ID NO: 195, SEQ ID NO: 226, SEQ ID NO: 270, SEQ ID NO: 311, SEQ ID NO: 29, SEQ ID NO: 21, SEQ ID NO: 42, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 9, SEQ ID NO: 266, SEQ ID NO: 317, SEQ ID NO: 25, SEQ ID NO: 233, SEQ ID NO: 62, SEQ ID NO: 20 and SEQ ID NO: 69. The predicted binding energy of the 27 antibody sequences was compared to their in-vitro binding energy. Table 9 shows a comparison of the predicted binding energy and binding energy obtained from surface plasmon resonance.Table 9. Predicted binding energy and in-vitro binding energy of antibodies with a common heavy chain and varying light chainExample 5: Method of Quantifying and Qualifying Binding of Representative Antibodies with TestosteroneSurface Plasmon Resonance

[0148] The 27 antibody sequences identified in Example 4 were produced and tested using surface plasmon resonance. 1 pg of each antibody was immobilized on a Cytiva CM1 gold sensor chip. A testosterone-BSA conjugate was used with phosphate buffer to visualize any significant change in the molecular mass. To estimate the Ka (association constant), 100 pL of 1 mg / mL testosterone-BSA was passed on the sensor chip at a flow rate of 50pL / minute for 3 minutes, and the response was measured. To estimate the Kd (dissociation constant), 1 M HC1 was passed on the sensor chip at a flow rate of 50 pL / minute for 3 minutes, and the response was measured.

[0149] Three antibodies had a binding affinity (KD) of < 13 kcal / mol, and two antibodies had a KD of > 10 kcal / mol. and 22 antibodies in between the range so that the performance of the in-silico system could be validated across the entire range of predicted binding affinities.

[0150] The AG was calculated using the following equation:AG = -RT In (Kd / Ka)

[0151] The average experimental AG was plotted against the predicted AG value (FIG. 4), yielding a R2value of 0.87. This indicates that the computation system described in Examples 1-3 can predict binding energy similar to experimental binding energy.

[0152] Additionally, the antibodies were checked on a lateral flow assay by immobilizing testosterone-BSA on a nitrocellulose (NC) membrane as a test line and checking for the binding of antibodies to the test line. In this assay, 1 mg / mL of testosterone-BSA was sprayed on the nitrocellulose membrane at 1 pL / cm and a spraying speed of 10 mm / second. The sprayed NC membrane was dried at 40 degrees for 15 minutes and then left overnight at room temperature. Candidate antibodies were covalently conjugated to europium chelates using EDC (l-ethyl-3- (3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) coupling. 1 pg / mL of the final antibody-europium conjugate was made to flow on the nitrocellulose. The results of the lateral flow assay are shown in FIG. 5.Example 6: Clustering of Amino Acid Sequences Based on Chemical Properties of Interacting Amino Acids

[0153] An algorithm to cluster the amino acid sequence in Table 2 based on their mutations. This clustering algorithm identified common mutation patterns that contribute to the effectiveness of an antibody. This understanding can guide optimization and refinement of sequences to select for favourable properties.

[0154] By grouping sequences with similar mutations, development efforts could be focused the most promising clusters. This targeted approach reduces time and resources spent on testing and validation.

[0155] Each protein sequence was defined by the amino acids at the mutated positions, e.g., 96, 99, 101, simplified to FDM, to focus on the key variations relevant for clustering.

[0156] The Hamming distance between these simplified sequences was computed to quantify their dissimilarity based on chemical mutations. The Hamming distance, a measure of the number of positions at which the corresponding symbols are different, was used to compute the dissimilarities between all pairs of sequences. For two sequences SI and S2, the Hamming distance is defined as:where 6 is the Kronecker delta which is 1 if si; s2j and 0 otherwise.

[0157] The dendrogram in FIG. 5 illustrates the hierarchical clustering process. Each leaf represents a protein sequence, and the height at which two leaves merge represents the distance between two clusters.

[0158] Using the Hamming distance matrix, hierarchical clustering was performed. The single-linkage method merges clusters based on the shortest distance between members, ensuring high intra-cluster similarity and distinct inter-cluster differences.

[0159] By applying a cutoff distance of 8.8, the antibodies were grouped into approximately 10 clusters. The resulting clusters offered a detailed view of the mutation landscape within the dataset. 1. Cluster 1 (SEQ ID NOs: 1-10):• Sequence Feature: Aromatic pocket with a mutation at position 101 facilitating hydrogen bond formation.2. Cluster 2 (SEQ ID NOs: 11-42):• Sequence Feature: Mutation at position 101 enabling 0-0 interaction with testosterone.3. Cluster 3 (SEQ ID NOs: 43-84):• Sequence Feature: Aromatic group at position 101.4. Cluster 4 (SEQ ID NOs: 85-94):• Sequence Feature: Acidic or highly flexible residue at position 99.5. Cluster 5 (SEQ ID NOs: 95-136):• Sequence Feature: Amine-containing group at position 96.6. Cluster 6 (SEQ ID NOs: 137-189):• Sequence Feature: Four-carbon-long R group at position 101 with a pKa close to 7.4.7. Cluster 7 (SEQ ID NOs: 190-224):• Sequence Feature: Methionine at position 101.8. Cluster 8 (SEQ ID NOs: 225-259):• Sequence Feature: Histidine at position 101.9. Cluster 9 (SEQ ID NOs: 260-190):• Sequence Feature: Glutamine at position 101.10. Cluster 10 (SEQ ID NOs: 291-319):• Sequence Feature: Arginine at position 101.Table 10. List of motifs bound to testosterone and interacting partners of LCDR3 region with testosteroneExample 7: Engineering and Evaluation of Antibody Variants for Salivary Hormone Detection

[0160] The antibodies in this example were generated from a consensus light chain and a 119 J (PDB: pdb_00001i9j) heavy chain (sequences listed in Table 11). The heavy chain remained unmodified, while optimization efforts focused on the light chain, particularly in its LCDR3 region. As explained above, molecular docking studies have shown that residues 96, 99, and 101 exhibit significant binding interactions with testosterone. Those residues and their surrounding residues were varied in the light chain sequence (94, 95, 96, 97, 98, 99, 100, 101,and 102). The goal was to maximizing sensitivity and analytical precision, in order to generate antibodies for hormone detection using lateral flow assays (LFAs).Table 11. Antibody sequences• Focus Region: CDR-L3 in the light chain

[0161] Four key salivary hormones (testosterone, estradiol, progesterone, and cortisol) were identified as the primary ligands for docking and binding affinity screening based on concentration data and relevance to LFA diagnostics.Cross-Reactivity and the Role of ABE Thresholding

[0162] In lateral flow assays (LFAs), cross-reactivity occurs when the antibody binds not only to the intended target analyte but also to structurally similar molecules. This compromises specificity and can lead to false positives, especially when detecting low-abundance hormones in complex biological fluids like saliva. Given the similarity amongst steroid hormone structures (e.g., testosterone, cortisol, estradiol, progesterone), designing antibodies with high selectivity for testosterone is particularly challenging. Even minor unintended binding events can skew results when hormone concentrations vary over narrow physiological ranges.

[0163] Accordingly, binding energy (ABE) thresholds derived from thermodynamic principles were used to control both sensitivity and selectivity. Specifically, a ABE threshold of -2.5 kcal / mol was initially adopted to isolate high-affinity binders. This corresponds to -10- fold higher binding affinity compared to a neutral baseline, enough to ensure LFA sensitivity.

[0164] For cortisol, theoretical calculations suggest that a ABE of -2.97 kcal / mol is needed for 5% CV precision and ABE of -2.26 kcal / mol is needed for 10% CV precision and.Strategic Implications for Assay Design

[0165] By applying a ABE cutoff of -2.56 kcal / mol, sequences were filtered out that either bind too weakly to the target (insufficient sensitivity) or bind comparably to multiple ligands (higher cross-reactivity risk). This strategy supports analytical precision while improving diagnostic reliability, particularly in multiplex or point-of-care settings where specificity is paramount.Table 12. Salivary hormone concentrations and binding requirements (CV < 5%)

[0166] Using the thermodynamic relationship:AG = -RT ln(Ka), and approximate at 298K:ABE « -0.592 x ln(Fold change in Ka)

[0167] Table 13 shows exemplary Ka increases and corresponding ABE for cortisol. Table 14 shows the corresponding ABE adjustment for a given CV variation.Table 13. Ka increase and ABE adjustmentTable 14. CV variation and ABE adjustmentMutation Strategy and Sequence Library Generation

[0168] Targeted mutation was performed at positions [96, 99, 101], [97, 100, 102] or [95, 98, 100] in light chain sequence (SEQ ID NO: 1). Each site was mutated with all 20 amino acids. This produced 8000 total sequences per three mutation combination. The heavy chain remained fixed.Docking and Sequence Filtering

[0169] All 8000 variants were docked with each of the 4 hormones (total docking events: 32,000). The docking energies and protein parameters were recorded. The following docking and parameter filters were used:Protein Parameters Computed:• Isoelectric Point (pl)• GRAVY• Instability Index• Molecular Weight• AromaticityPrimary criteria:• Instability Index < 40• pl: 5.5-8.5

[0170] Only 49 light chain sequences passed both docking and parameter filters listed above. These sequences are listed in Table 6 above. The binding affinities are listed in Table 15. After expanding pl range to 4.5-9.5, 113 light chain sequences met the relaxed criteria. The additional sequences that met these criteria are listed in Table 7 above.Table 15. Binding affinities

Claims

CLAIMS1. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.

2. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

3. The anti-testosterone antibody or antigen-binding fragment of claim 2, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 349, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

4. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs), and wherein LCDR3 comprises the amino acid sequence of any one of claims 1 to3,5. The anti-testosterone antibody or antigen -binding fragment of any one of claims 1 to4, wherein the LCDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 473-595.

6. The anti-testosterone antibody or antigen-binding fragment of any one of claims 1 to5, wherein the antibody or antigen-binding fragment comprises three HCDRs and threeLCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 321, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 322, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 323, as defined by the IMGT numbering system.

7. The anti-testosterone antibody or antigen-binding fragment of claim 6, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320.

8. The anti-testosterone antibody or antigen-binding fragment of claim 6, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system.

9. The anti-testosterone antibody or antigen-binding fragment of claim 6, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and an amino acid sequence selected from any one of SEQ ID NOs: 326-348 (LCDR3), as defined by the IMGT numbering system.

10. The anti-testosterone antibody or antigen -binding fragment of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, as defined by the IMGT numbering system.

11. The anti-testosterone antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463.

12. The anti-testosterone antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 349 (LCDR3), as defined by the IMGT numbering system.

13. The anti-testosterone antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 473-595 (LCDR3), as defined by the IMGT numbering system.

14. The anti-testosterone antibody or antigen-binding fragment of any one of claims 1 to13, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 350.

15. The anti-testosterone antibody or antigen -binding fragment of any one of claims 1 to14, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-319 or SEQ ID NOs: 351-462.

16. The anti-testosterone antibody or antigen-binding fragment of any one of claims 1, 4, 5 to 9, 14, or 15, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid of SEQ ID NO: 350, wherein X1is F, L, Q, H, T, N, R, or M; X2= T, S, D, G, H, Y, N, R, or Q; and X3= T, F, N, M, L, H, R, or Q.Ill17. The anti-testo sterone antibody or antigen -binding fragment of any one of claims 2 to 4 and 10 to 15, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, D, E, I, L, or V; X2= H, E, or P; and X3= E, L, Y, L, F, or N.

18. The anti-testo sterone antibody or antigen-binding fragment of any one of claims 2 to 4 and 10 to 15, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 350, wherein X1is A, C, D, E, F, G, H, I, L, N, P, T, V, W, or Y; X2= F, H, N, P, Q, R, W, I, M, C, T, E, S, L, or A; and X3= K, L, E, F, Y, H, I, N, M, G, W, A, R, or D.

19. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 464, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

20. The anti-testosterone antibody or antigen-binding fragment thereof of claim 19, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 464, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

21. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs), and wherein LCDR3 comprises the amino acid sequence of claim 19 or claim 20.

22. The anti-testosterone antibody or antigen-binding fragment of any one of claims 19 to 21, wherein the LCDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 351-462.

23. The anti -testosterone antibody or antigen -binding fragment of any one of claims 19 to 22, wherein the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, as defined by the IMGT numbering system.

24. The anti-testosterone antibody or antigen-binding fragment of claim 23, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463.

25. The anti-testosterone antibody or antigen-binding fragment of claim 23, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 464 (LCDR3), as defined by the IMGT numbering system.

26. The anti-testosterone antibody or antigen-binding fragment of claim 23, wherein the antibody or antigen-binding fragment comprises wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and an amino acid sequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 351-462 (LCDR3), as defined by the IMGT numbering system.

27. The anti-testosterone antibody or antigen -binding fragment of any one of claims 19 to 26, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 466.

28. The anti-testosterone antibody or antigen-binding fragment of any one of claims 19 to27, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from any one of SEQ ID NOs: 1-319 or SEQ ID NOs: 351-462.

29. The anti-testosterone antibody or antigen-binding fragment of any one of claims 19 to28, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is C, D, E, H, I, or W; X5= M, I, Q, W, A, S, or E; and X6= E, H, T, C, Y, D, or G.

30. The anti-testosterone antibody or antigen-binding fragment of any one of claims 19 to29, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 466, wherein X4is W, E, D, C, I, or H; X5= E, W, Q, I, M, S, or A; and X6= H, Y, T, C, E, G, D, L, F, or I.

31. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 465, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

32. The anti-testosterone antibody or antigen-binding fragment thereof of claim 31, wherein the antibody or antigen-binding fragment binds specifically to testosterone, and wherein the antibody or antigen-binding fragment comprises an amino acid sequence of SEQ ID NO: 465, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

33. An anti-testosterone antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs), and wherein LCDR3 comprises the amino acid sequence of claim 31 or claim 32.

34. The anti-testosterone antibody or antigen-binding fragment of any one of claims 31 to33, wherein the LCDR3 comprises an amino acid sequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 351-462.

35. The anti-testosterone antibody or antigen-binding fragment of any one of claims 31 to34, wherein the antibody or antigen-binding fragment comprises three HCDRs and three LCDRs, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 468, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 469, and the HCDR3 comprises the amino acid sequence of SEQ ID NO: 470, as defined by the IMGT numbering system.

36. The anti-testosterone antibody or antigen-binding fragment of claim 35, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463.

37. The anti-testosterone antibody or antigen-binding fragment of claim 35, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), and SEQ ID NO: 465 (LCDR3), as defined by the IMGT numbering system.

38. The anti-testosterone antibody or antigen-binding fragment of claim 35, wherein the antibody or antigen-binding fragment comprises three HCDRs comprising amino acid sequences of SEQ ID NO: 468 (HCDR1), SEQ ID NO: 469 (HCDR2), and SEQ ID NO: 470 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1),SEQ ID NO: 325 (LCDR2), and an amino acid sequence selected from any one of SEQ ID NOs: 326-348 or SEQ ID NOs: 351-462 (LCDR3), as defined by the IMGT numbering system.

39. The anti-testosterone antibody or antigen-binding fragment of any one of claims 31 to38, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence of SEQ ID NO: 467.

40. The anti-testosterone antibody or antigen-binding fragment of any one of claims 31 to39, wherein the antibody or antigen-binding fragment comprises a light chain comprising an amino acid sequence selected from any one of SEQ ID NO: 1-319 or SEQ ID NOs: 351-462.

41. The anti-testosterone antibody or antigen-binding fragment of any one of claims 31 to40, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is F, T, D, E, G, K, L, V Y, Q, or N; X8= F, T, D, E, G, K, L, V, Y, Q, or N; and X9= C, D, E, F, G, H, I, L, M, N, Q, V, or Y.

42. The anti-testosterone antibody or antigen-binding fragment of any one of claims 31 to41, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence of SEQ ID NO: 467, wherein X7is T, F, D, E, G, K, L, V, Y, Q, or N; X8= Y, T, C, E, G, D, L, F, or I; and X9= Y, C, D, E, F, G, H, I, L, M, N, Q, or V.

43. The anti-testosterone antibody or antigen -binding fragment of any one of claims 1 to 5 and 10 to 42, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 463 and a light chain comprising an amino acid sequence selected from SEQ ID NO: 1-319 or SEQ ID NOs: 351-462.

44. The anti-testosterone antibody or antigen-binding fragment of any one of claims 1, 4 to 9, and 16, wherein the antibody or antigen -binding fragment comprises a heavy chaincomprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1-319.

45. The anti -testosterone antibody or antigen-binding fragment of claim 44, wherein the antibody or antigen-binding fragment comprises:(i) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:326 (LCDR3), as defined by the IMGT numbering system;(ii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:327 (LCDR3), as defined by the IMGT numbering system;(iii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:328 (LCDR3), as defined by the IMGT numbering system;(iv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:329 (LCDR3), as defined by the IMGT numbering system;(v) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:330 (LCDR3), as defined by the IMGT numbering system;(vi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:331 (LCDR3), as defined by the IMGT numbering system;(vii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprisingamino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:332 (LCDR3), as defined by the IMGT numbering system;(viii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:333 (LCDR3), as defined by the IMGT numbering system;(ix) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:334 (LCDR3), as defined by the IMGT numbering system;(x) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:335 (LCDR3), as defined by the IMGT numbering system;(xi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:336 (LCDR3), as defined by the IMGT numbering system;(xii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:337 (LCDR3), as defined by the IMGT numbering system;(xiii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:338 (LCDR3), as defined by the IMGT numbering system;(xiv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:339 (LCDR3), as defined by the IMGT numbering system;(xv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:340 (LCDR3), as defined by the IMGT numbering system;(xvi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:341 (LCDR3), as defined by the IMGT numbering system;(xvii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:342 (LCDR3), as defined by the IMGT numbering system;(xviii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:343 (LCDR3), as defined by the IMGT numbering system;(xix) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:344 (LCDR3), as defined by the IMGT numbering system;(xx) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:345 (LCDR3), as defined by the IMGT numbering system;(xxi) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:346 (LCDR3), as defined by the IMGT numbering system;(xxii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprisingamino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:347 (LCDR3), as defined by the IMGT numbering system;(xxiii) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:348 (LCDR3), as defined by the IMGT numbering system; or(xxiv) three HCDRs comprising amino acid sequences of SEQ ID NO: 321 (HCDR1), SEQ ID NO: 322 (HCDR2), and SEQ ID NO: 323 (HCDR3); and three LCDRs comprising amino acid sequences of SEQ ID NO: 324 (LCDR1), SEQ ID NO: 325 (LCDR2), SEQ ID NO:349 (LCDR3), as defined by the IMGT numbering system.

46. The anti-testosterone antibody or antigen-binding fragment of claim 45, wherein the antibody or antigen-binding fragment comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence selected from SEQ ID NOs: 1, 5, 9, 12, 14, 16, 20, 21, 25, 29, 42, 54, 62, 69, 87, 106, 140, 195, 226, 233, 266, 270, 311, and 317.

47. The anti-testosterone antibody or antigen-binding fragment of claim 45 or claim 46, wherein the antibody or antigen-binding fragment comprises:(i) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 5;(ii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 9;(Hi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 12;(iv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 14;(v) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 16;(vi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 20;(vii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 21;(viii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 25;(ix) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 29;(x) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 42;(xi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 54;(xii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 62;(xiii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 69;(xiv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 87;(xv) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 106;(xvi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 140;(xvii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 195;(xviii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 226;(xix) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 270;(xx) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 311;(xxi) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 317; or(xxii) a heavy chain comprising an amino acid sequence of SEQ ID NO: 320 and a light chain comprising an amino acid sequence of SEQ ID NO: 1.

48. An antibody or antigen-binding fragment that competes for binding to testosterone with the antibody or antigen-binding fragment of any one of claims 1 to 47.

49. An antibody or antigen-binding fragment of any one of claims 1 to 48 that binds to the ketone group at the 3-position of testosterone and / or the hydroxyl group at the 17-position of testosterone.

50. A method of treating a subject in need of inhibition of testosterone, comprising administering to the patient a therapeutically effective amount of the antibody or antigenbinding fragment of any one of claims 1 to 49.

51. A method of detecting testosterone in a patient, comprising providing a biological sample from the patient and contacting the biological sample with the antibody or antigenbinding fragment of any one of claims 1 to 49, wherein the antibody or antigen-binding fragment binds to testosterone and thereby detects the presence of testosterone in the sample.

52. The method of claim 51, wherein the antibody or antigen-binding fragment comprises a detectable label.

53. The method of claim 51, wherein the detection method comprises an enzyme-linked immunosorbent assay (ELISA).

54. The method of claim 51 , wherein the detection method comprises a chemiluminescent immunoassay (CLIA).

55. The method of claim 51, wherein the detection method comprises a radioimmunoassay (RIA).

56. The method of claim 51, wherein the detection method comprises a coimmunoprecipitation assay (Co-IP).

57. The method of claim 51, wherein the detection method comprises a lateral flow assay (LFA).

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