RNA compositions targeting HIV

Abstract

The present disclosure provides compositions (e.g., pharmaceutical compositions) for delivering anti-HIV antibody agents and related techniques (e.g., components thereof and / or methods related thereto). In addition to this, the present disclosure also provides polyribonucleotides encoding an immunoglobulin chain of an anti-HIV antibody agent.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 63 / 477,460, filed December 28, 2022, and U.S. Provisional Application No. 63 / 517,542, filed August 3, 2023, which are incorporated herein by reference in their entirety. Background Technology

[0003] Human immunodeficiency virus (HIV) is an infectious virus associated with acquired immunodeficiency syndrome (AIDS). According to the World Health Organization, more than 37 million people worldwide are currently living with HIV. In 2020, approximately 680,000 people died from HIV-related causes, and about 1.5 million people contracted HIV. Currently, there is no cure for HIV. Summary of the Invention

[0004] This disclosure recognizes that HIV mutates rapidly. The high variability of HIV allows it to evade host immunity and / or treatment. To combat viral escape, combination therapies targeting HIV, including anti-HIV antibody agents, have been considered. However, the development of such combination therapies is hampered by several challenges. First, developing individual HIV therapies is both time-consuming and expensive. For example, the development of anti-HIV antibodies faces challenges related to demanding and costly production, including purification and formulation methods associated with protein therapeutics. Second, combination therapies present regulatory challenges. In addition to ensuring the safety and efficacy of combination therapies, stringent regulations governing the manufacture of individual therapies, the mixing of multiple therapies, and quality control during storage and administration complicate their use. Third, administering antibodies to subjects can be painful and time-consuming. Generally, antibodies are administered intravenously over a prolonged period. The administration of multiple antibodies increases the complexity of antibody administration, thereby increasing patient discomfort and consuming additional time. Finally, recombinant antibodies may have a short serum half-life.

[0005] This disclosure provides insights into addressing these challenges, making it possible not only to safely, reliably, and potently deliver a single anti-HIV antibody to a subject, but also to deliver multiple anti-HIV therapeutics, including multiple anti-HIV antibody agents. For example, this disclosure describes antibody agents or portions thereof (e.g., immunoglobulin chains) delivered to a subject via polynucleotides. Antibody agents delivered to a subject as one or more polynucleotides encoding antibody agents are referred to herein as “RiboMab”. Following delivery of one or more polynucleotides encoding antibody agents to a subject, the subject’s body expresses the antibody agent, i.e., “RiboMab”. Furthermore, the term “RibobNAb” refers to RiboMab containing all or part of a broad-spectrum neutralizing antibody (bNAb), such as a broad-spectrum neutralizing antibody targeting HIV. Utilizing polynucleotides as therapeutic agents (in contrast to administering antibody agents themselves) involves simpler and less expensive manufacturing processes. The production of polynucleotides encoding antibody agents (e.g., anti-HIV antibody agents) is less complex and can simplify manufacturing (e.g., by circumventing the need for intensive glycan production and analysis), thereby mitigating the regulatory and manufacturing challenges associated with the development and use of the antibody agents themselves. Furthermore, polynucleotides efficiently produce effects similar to recombinant proteins but often require much smaller doses to be administered to subjects. This is because the polynucleotide encoding, for example, an anti-HIV antibody agent can be administered to the subject, and the subject's body itself produces the anti-HIV antibody agent. Using smaller doses can provide a more pleasant experience for patients and increase patient adherence to treatment regimens. This disclosure also provides techniques for addressing certain limitations of recombinant antibody technology by utilizing RNA technology as a means of directly expressing antibody agents in the patient's cells, limitations including, for example, the short serum half-life of recombinant antibodies.

[0006] RiboMab technology also allows for the simultaneous administration of two or more antibody agents to a subject. Typically, antibodies produced by, for example, humans, consist of four polypeptide chains—two “heavy” chains and two “light” chains. Each polypeptide chain (whether heavy or light) includes (1) a “variable” domain whose sequence varies between antibodies and whose structure determines the antigen the antibody binds to, and (2) a “constant” domain whose sequence and structure generally remain unchanged in a given class of antibodies, thus having little effect on antigen binding. In humans, specialized white blood cells, “B cells,” produce antibodies. The heavy and light chains assemble to form antibodies through two key pairings: (1) fragment crystallizable (Fc) domains of the two heavy chains pair together, and (2) each of the two light chains pairs with one heavy chain via a disulfide bond. In normal antibody production in humans, a single B cell produces a single antibody. In this case, the correct pairing of the heavy and light chains is ensured because only one heavy chain and one light chain are present in each B cell. However, administering a nucleic acid composition encoding more than one antibody to a subject requires proper assembly of the immunoglobulin chains (e.g., heavy and light chains) to avoid the formation of unwanted byproducts (e.g., antibody agents with unintended pairings).

[0007] In addition, this disclosure also provides polynucleotides encoding immunoglobulin chains of antibody agents.

[0008] On one hand, this disclosure provides a polynucleotide encoding an immunoglobulin chain for an antibody agent, wherein the immunoglobulin chain comprises a single-chain variable fragment (scFv), and the scFv comprises a heavy chain variable (VH) domain, a linker, and a light chain variable (VL) domain, wherein the VH domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences according to SEQ ID NO:18, 21, and 24, respectively, and wherein the VH domain comprises a first mutation at residue 3 and a second mutation at residue 5 relative to the amino acid sequence according to SEQ ID NO:1494; and wherein the VL domain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise amino acid sequences according to SEQ ID NO:27, 30, and 33, respectively.

[0009] In some embodiments, the first mutation comprises a substitution mutation resulting in a positively charged amino acid at residue 3. In some embodiments, the positively charged amino acid comprises an amino acid selected from Lys(K), Arg(R), or His(H). In some embodiments, the positively charged amino acid comprises a His(H) residue. In some embodiments, the second mutation comprises a substitution mutation resulting in a polar amino acid at residue 5. In some embodiments, the polar amino acid comprises an amino acid selected from Ser(S), Thr(T), Tyr(Y), Asn(N), or Gln(Q). In some embodiments, the polar amino acid comprises a Thr(T) residue. In some embodiments, the VH domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO:36. In some embodiments, the VH domain comprises the amino acid sequence SEQ ID NO:1494, having the substitution mutations Q3H and V5T. In some embodiments, the VH domain comprises an amino acid sequence according to SEQ ID NO:36.

[0010] In some embodiments, the VL domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence according to SEQ ID NO:43. In some embodiments, the VL domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO:43. In some embodiments, the VL domain comprises the amino acid sequence according to SEQ ID NO:43.

[0011] In some implementations, the scFv sequentially comprises: (i) the VH structural domain, (ii) the connector, and (iii) the VL structural domain.

[0012] In some implementations, the scFv sequentially comprises: (i) the VL structural domain, (ii) the connector, and (iii) the VH structural domain.

[0013] In some embodiments, the linker comprises the amino acid sequence according to SEQ ID NO:48. In some embodiments, the linker comprises the amino acid sequence according to SEQ ID NO:59. In some embodiments, the immunoglobulin chain further comprises a second linker following the scFv domain. In some embodiments, the scFv and the second linker comprise or consist of the amino acid sequence according to SEQ ID NO:64, 67, 70, or 73. In some embodiments, the immunoglobulin chain comprises a hinge domain following the second linker. In some embodiments, the hinge domain comprises or consists of the amino acid sequence according to SEQ ID NO:167.

[0014] In some embodiments, the immunoglobulin chain comprises one or more constant domains, and wherein the scFv is operatively linked to the one or more constant domains. In some embodiments, the hinge domain is between the scFv and the one or more constant domains. In some embodiments, the one or more constant domains comprise a CH3 domain. In some embodiments, the CH3 domain comprises a G1m17,1 or G1m3 allotype. In some embodiments, the CH3 domain comprises one or more substitution mutations, wherein the one or more substitution mutations comprise or consist of M88L, N94S, or a combination thereof, and wherein the substitution mutation position is relative to the amino acid sequence according to SEQ ID NO:1495. In some embodiments, the CH3 domain comprises substitution mutations at residues 16 and 18 relative to the amino acid sequence according to SEQ ID NO:1495. In some embodiments, the CH3 domain comprises the amino acid sequence SEQ ID NO:1495, having substitution mutations D16E and L18M. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:116.

[0015] In some embodiments, the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1343. In some embodiments, the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1346. In some embodiments, the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1349. In some embodiments, the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1352.

[0016] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a secretion signal. In some embodiments, the secretion signal comprises a ribonucleic acid sequence that is at least 90% identical to SEQ ID NO:4 or 8. In some embodiments, the secretion signal comprises a ribonucleic acid sequence comprising SEQ ID NO:4 or 8.

[0017] In some embodiments, the polynucleotide comprises one or more non-coding sequence elements. In some embodiments, the one or more non-coding sequence elements enhance RNA stability and / or translation efficiency. In some embodiments, the one or more non-coding sequence elements comprise a 3' untranslated region (UTR), a 5' UTR, a 5'-cap, a polyadenine (polyA) tail, or any combination thereof. In some embodiments, the polyA tail is or comprises a modified polyA sequence, preferably a discontinuous polyA tail. In some embodiments, the polyA tail comprises or consists of a sequence at least 90% identical to or constitutes the sequence of SEQ ID NO:16. In some embodiments, the 3' UTR comprises or consists of a nucleic acid sequence at least 90% identical to or constitutes the sequence of SEQ ID NO:14. In some embodiments, the 5' UTR comprises or consists of a nucleic acid sequence at least 90% identical to or constitutes the sequence of SEQ ID NO:10. In some embodiments, the 5'-cap is (m27,3'-O)Gppp(m2'-O)ApG. In some embodiments, the polynucleotide comprises one or more modified ribonucleotides. In some embodiments, the one or more modified ribonucleotides comprise pseudouridine.

[0018] In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1000. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1048. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1096. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1144.

[0019] On the other hand, this disclosure provides a polynucleotide comprising or composed of a ribonucleic acid sequence according to any one of SEQ ID NO: 1000, 1048, 1096 or 1044.

[0020] On one hand, this disclosure also provides a composition comprising one or more polynucleotides described herein. In some embodiments, the composition further comprises lipid nanoparticles, polymeric complexes (PLX), lipotropic polymeric complexes (LPLX), or liposomes, wherein one or more polynucleotides are wholly or partially encapsulated within the lipid nanoparticles, polymeric complexes (PLX), lipotropic polymeric complexes (LPLX), or liposomes. In some embodiments, the composition further comprises lipid nanoparticles, wherein one or more polynucleotides are encapsulated within the lipid nanoparticles. In some embodiments, the lipid nanoparticles are cationic lipid nanoparticles.

[0021] On the other hand, this disclosure provides a pharmaceutical composition comprising the composition as described in any one of claims 53-56 and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is used for the treatment and / or prevention of HIV, the treatment and / or prevention comprising administering the pharmaceutical composition to a subject.

[0022] On the other hand, this disclosure provides a method comprising administering the pharmaceutical composition described herein to a subject.

[0023] In some embodiments, administration of the pharmaceutical composition to a subject results in the expression of an antibody agent in the subject. In some embodiments, the antibody agent is expressed in the subject at titers of: (a) at least 1 μg / ml in plasma; or (b) at least 1 μg / ml in serum. In some embodiments, the antibody agent is expressed in the subject at titers of: (a) at least 10 μg / ml in plasma; or (b) at least 10 μg / ml in serum. In some embodiments, the antibody agent can be detected in the subject's serum for a period of at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, or at least 30 days. In some embodiments, the antibody agent can be detected in the subject's serum for a period of at least 30 days.

[0024] In some embodiments, the antibody agent is capable of neutralizing one or more HIV strains when tested in a TZM-bl cell pseudovirus neutralization assay at antibody agent concentrations up to 25 μg / ml. In some embodiments, the antibody agent delivered in polynucleotide form has higher neutralizing activity against one or more HIV strains compared to an equivalent amount of parental control antibody delivered in polynucleotide form, wherein the parental control antibody is an IgG antibody containing the same VH and VL domains as the antibody agent. In some embodiments, the one or more HIV strains comprise one or more HIV strains selected from the following: ZM53M.PB12, Du156.12, Q769.d22, 0330.v4.c3, R2184.c04, 89-F1_2_25, CAP204_2_00_F6_6, Ce1176_A3, 6980.v0.c31, PVO.4, CAP45, CNE8, T250-4, 3103.v3.c10, and C1080_c3.

[0025] In some implementations, the subjects are HIV-infected or at risk of developing HSV infection.

[0026] In some implementations, the method is a method for treating and / or preventing HIV infection.

[0027] In some embodiments, this disclosure also provides for the use of the compositions described herein or the pharmaceutical compositions described herein for the treatment and / or prevention of HIV in a subject.

[0028] In some embodiments, this disclosure provides a method for generating an antibody agent, the method comprising administering the composition or pharmaceutical composition described herein to cells, causing the cells to express and secrete the antibody agent. In some embodiments, the cells are in a subject and the antibody agent is generated at a treatment-related plasma concentration or treatment-related serum concentration. In some embodiments, the treatment-related plasma concentration or treatment-related serum concentration is at least 10 μg / ml.

[0029] On one hand, the immunoglobulin chain includes a heavy chain variable (VH) domain. In some embodiments, the VH domain includes a heavy chain complementarity-determining region (HCDR)1, which contains the amino acid sequence according to SEQ ID NO:18; HCDR2, which contains the amino acid sequence according to SEQ ID NO:21; and HCDR3, which contains the amino acid sequence according to SEQ ID NO:24.

[0030] In some embodiments, the VH domain comprises or consists of the amino acid sequence according to SEQ ID NO:24. In some embodiments, the VH domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:36.

[0031] In some embodiments, the polynucleotide comprises a VH domain coding sequence. In some embodiments, the VH domain coding sequence comprises (a) an HCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:19, (b) an HCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:22, and (c) an HCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:25.

[0032] In some embodiments, the VH domain coding sequence comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:37. In some embodiments, the VH domain coding sequence comprises or consists of the ribonucleic acid sequence according to SEQ ID NO:37. In some embodiments, the VH domain coding sequence comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:39. In some embodiments, the VH domain coding sequence comprises or consists of the ribonucleic acid sequence according to SEQ ID NO:41. In some embodiments, the VH domain coding sequence comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:39. In some embodiments, the VH domain coding sequence comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:39.

[0033] In some embodiments, the immunoglobulin chain containing the VH domain as described herein includes one or more constant domains. In some embodiments, the VH domain is operatively linked to one or more constant domains.

[0034] In some embodiments, the immunoglobulin chain containing the VH domain as described herein includes one or more constant domains. In some embodiments, the VH domain is operatively linked to one or more constant domains.

[0035] In some embodiments, one or more constant domains comprise a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain.

[0036] In some embodiments, one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH3 domain.

[0037] In some embodiments, one or more constant domains comprise hinge domains. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the hinge domain.

[0038] In some embodiments, one or more constant domains comprise the CH1 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH1 domain.

[0039] In some embodiments, one or more constant domains comprise a CL domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CL domain.

[0040] In some implementations, the immunoglobulin chain containing the VH domain as described herein includes a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain.

[0041] In some implementations, the immunoglobulin chain containing the VH domain as described herein includes a CL domain, a hinge domain, a CH2 domain, and a CH3 domain.

[0042] This disclosure also provides a polynucleotide encoding an immunoglobulin chain for an antibody agent, wherein the immunoglobulin chain comprises a light chain variable (VL) domain.

[0043] In some embodiments, the VL domain includes (a) a light chain complementarity-determining region (LCDR) 1, which contains the amino acid sequence according to SEQ ID NO:27; (b) LCDR 2, which contains the amino acid sequence according to SEQ ID NO:30; and (c) LCDR 3, which contains the amino acid sequence according to SEQ ID NO:33.

[0044] In some embodiments, the polynucleotide comprises a VL domain coding sequence. In some embodiments, the VL domain coding sequence comprises (a) an LCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:28; an LCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:31; and an LCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:34.

[0045] In some embodiments, the VL domain comprises or is composed of the amino acid sequence according to SEQ ID NO:43. In some embodiments, the VL domain coding sequence comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:44. In some embodiments, the VL domain coding sequence comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:46.

[0046] In some embodiments, the immunoglobulin chain containing the VL domain also includes a constant domain. In some embodiments, the VL domain is operatively linked to the constant domain. In some embodiments, the polynucleotide contains a ribonucleic acid sequence encoding the constant domain.

[0047] In some embodiments, the immunoglobulin chain containing the VL domain also contains a CL domain. In some embodiments, the polynucleotide contains a ribonucleic acid sequence encoding the CL domain. In some embodiments, the CL domain is a κ constant domain. In some embodiments, the polynucleotide contains a ribonucleic acid sequence encoding a light chain constant domain.

[0048] In some embodiments, the immunoglobulin chain containing the VL domain also contains the CH1 domain. In some embodiments, the polynucleotide contains a ribonucleic acid sequence encoding the CH1 domain.

[0049] In addition, this disclosure also provides a polynucleotide encoding an immunoglobulin chain for an antibody agent, wherein the immunoglobulin chain comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain. In some embodiments, the VH domain comprises HCDR1, which comprises the amino acid sequence according to SEQ ID NO:18; HCDR2, which comprises the amino acid sequence according to SE QID NO:21; and HCDR3, which comprises the amino acid sequence according to SEQ ID NO:24. In some embodiments, the VL domain comprises LCDR1, which comprises the amino acid sequence according to SEQ ID NO:27; LCDR2, which comprises the amino acid sequence according to SEQ ID NO:30; and LCDR3, which comprises the amino acid sequence according to SEQ ID NO:33.

[0050] In some embodiments, the polynucleotide comprises a VH domain coding sequence and a VL domain coding sequence. In some embodiments, the VH domain coding sequence comprises an HCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:19; an HCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:22; and an HCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:25. In some embodiments, the VL domain coding sequence comprises an LCDR1 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:28; an LCDR2 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:31; and an LCDR3 coding sequence comprising or consisting of the ribonucleic acid sequence according to SEQ ID NO:34.

[0051] In some embodiments, the immunoglobulin chain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a VH domain, a linker, and a VL domain.

[0052] In some embodiments, the scFv sequentially comprises a VH domain, a linker, and a VL domain. In some embodiments, the scFv sequentially comprises a VH domain containing or consisting of the amino acid sequence according to SEQ ID NO:36; a linker; and a VL domain containing or consisting of the amino acid sequence according to SEQ ID NO:43.

[0053] In some embodiments, the scFv sequentially comprises a VL domain, a linker, and a VH domain. In some embodiments, the scFv sequentially comprises a VL domain containing or consisting of the amino acid sequence according to SEQ ID NO:43; a linker; and a VH domain containing or consisting of the amino acid sequence according to SEQ ID NO:36.

[0054] In some embodiments, the adapter comprises an amino acid sequence according to SEQ ID NO:48. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the adapter and comprises or consists of a sequence according to SEQ ID NO:49, 51, 53, 55, or 57.

[0055] In some embodiments, the adapter comprises an amino acid sequence according to SEQ ID NO:59. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the adapter and comprises or consists of a sequence according to SEQ ID NO:60 or 62.

[0056] In some embodiments, the immunoglobulin chain comprising the VH and VL domains as described herein includes one or more constant domains. In some embodiments, the VH and VL domains are operatively linked to one or more constant domains.

[0057] In some embodiments, the immunoglobulin chain comprises one or more constant domains, and a hinge domain is located between the scFv and the one or more constant domains.

[0058] In some embodiments, one or more constant domains comprise a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH2 domain.

[0059] In some embodiments, one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH3 domain.

[0060] In some embodiments, one or more constant domains comprise hinge domains. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the hinge domain.

[0061] In some embodiments, one or more constant domains comprise the CH1 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH1 domain.

[0062] According to this disclosure, the CH2 domain of any of the above embodiments may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:53. In some embodiments, the CH2 domain contains or is composed of the amino acid sequence according to SEQ ID NO:53.

[0063] In some embodiments, the ribonucleic acid sequence encoding the CH2 domain comprises a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:96. In some embodiments, the ribonucleic acid sequence encoding the CH2 domain comprises or consists of the sequence according to SEQ ID NO:96.

[0064] In some embodiments, the CH2 domain contains one or more substitution mutations. In some embodiments, one or more substitution mutations in the CH2 domain contain G236A, A330L, I332E, or combinations thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain contain G236A or are thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain contain G236A and I332E or are thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH2 domain contain G236A, A330L, and I332E or are thereof, and wherein the substitution mutation position is according to EU numbering.

[0065] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:56. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO:99. In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:102. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO:102.

[0066] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain, the ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:100. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and comprising or consisting of the sequence according to SEQ ID NO:100. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain, the ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:103. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence that encodes a CH2 domain and comprises or consists of a sequence according to SEQ ID NO: 103.

[0067] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:104. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO:104.

[0068] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:105. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and comprising or consisting of the sequence according to SEQ ID NO:105.

[0069] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:107. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO:107.

[0070] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:108. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and comprising or consisting of the sequence according to SEQ ID NO:108.

[0071] In some embodiments, the CH2 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:110. In some embodiments, the CH2 domain comprises or consists of the amino acid sequence according to SEQ ID NO:110.

[0072] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:111. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain and comprising or consisting of the sequence according to SEQ ID NO:111.

[0073] According to this disclosure, the CH3 domain of any of the above embodiments may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:113. In some embodiments, the CH3 domain contains or is composed of the amino acid sequence according to SEQ ID NO:113.

[0074] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:114. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprises or consists of the sequence according to SEQ ID NO:114.

[0075] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:131. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:131.

[0076] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:132. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprising or consisting of the sequence according to SEQ ID NO:132.

[0077] In some embodiments, the CH3 domain contains one or more substitution mutations. In some embodiments, the one or more substitution mutations in the CH3 domain contain M428L, N434S, or a combination thereof, or consist of such mutations, and wherein the substitution mutation position is according to an EU number.

[0078] In some embodiments, the CH3 domain comprises or is composed of the amino acid sequence according to SEQ ID NO:116. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding the CH3 domain and comprises or is composed of the sequence according to SEQ ID NO:117.

[0079] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:134. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:134.

[0080] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:135. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprises or consists of the sequence according to SEQ ID NO:135.

[0081] In some embodiments, one or more substitution mutations in the CH3 domain comprise Y349C, T366S, L368A, Y407V, or combinations thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise Y349C, T366S, L368A, Y407V, M428L, N434S, or combinations thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise Y349C, T366S, L368A, Y407V, M428L, and N434S, or combinations thereof, and wherein the substitution mutation position is according to EU numbering.

[0082] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:122. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:122.

[0083] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:123. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprising or consisting of the sequence according to SEQ ID NO:123.

[0084] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:140. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:140.

[0085] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:141. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprises or consists of the sequence according to SEQ ID NO:141.

[0086] In some embodiments, one or more substitution mutations in the CH3 domain comprise S354C, T366W, or a combination thereof, or constitute thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise S354C, T366W, M428L, N434S, or a combination thereof, or constitute thereof, and wherein the substitution mutation position is according to EU numbering. In some embodiments, one or more substitution mutations in the CH3 domain comprise S354C, T366W, M428L, and N434S, or constitute thereof, and wherein the substitution mutation position is according to EU numbering.

[0087] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:128. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:128.

[0088] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:129. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprising or consisting of the sequence according to SEQ ID NO:129.

[0089] In some embodiments, the CH3 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:146. In some embodiments, the CH3 domain comprises or consists of the amino acid sequence according to SEQ ID NO:146.

[0090] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:147. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain and comprising or consisting of the sequence according to SEQ ID NO:147.

[0091] According to this disclosure, the hinge domain of any of the above embodiments may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:161. In some embodiments, the hinge domain contains or is composed of the amino acid sequence according to SEQ ID NO:161.

[0092] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a hinge domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:162. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a hinge domain and comprising or consisting of the sequence according to SEQ ID NO:162.

[0093] In some embodiments, the hinge domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:164. In some embodiments, the hinge domain comprises or is composed of the amino acid sequence according to SEQ ID NO:164.

[0094] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a hinge domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:165. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a hinge domain and comprising or consisting of the sequence according to SEQ ID NO:165.

[0095] In some embodiments, the hinge domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:167. In some embodiments, the hinge domain comprises or is composed of the amino acid sequence according to SEQ ID NO:167.

[0096] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a hinge domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:168. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a hinge domain and comprising or consisting of the sequence according to SEQ ID NO:168.

[0097] According to this disclosure, the CH1 domain of any of the above embodiments may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:76. In some embodiments, the CH1 domain contains or is composed of the amino acid sequence according to SEQ ID NO:76.

[0098] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:77. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and comprising or consisting of the sequence according to SEQ ID NO:77.

[0099] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:81. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO:81.

[0100] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:82. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and comprising or consisting of the sequence according to SEQ ID NO:82.

[0101] In some embodiments, the CH1 domain contains one or more substitution mutations. In some embodiments, one or more substitution mutations in the CH1 domain contain K147E, K213D, or a combination thereof, or consist of such mutations, and wherein the substitution mutation position is according to an EU number.

[0102] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:84. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO:84.

[0103] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:85. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and comprising or consisting of the sequence according to SEQ ID NO:85.

[0104] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:76. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO:76.

[0105] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:77. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and comprising or consisting of the sequence according to SEQ ID NO:77.

[0106] In some embodiments, the CH1 domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:81. In some embodiments, the CH1 domain comprises or consists of the amino acid sequence according to SEQ ID NO:81.

[0107] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:82. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain and comprising or consisting of the sequence according to SEQ ID NO:82.

[0108] According to this disclosure, the CL domain of any of the above embodiments may contain an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:149. In some embodiments, the CL domain of any of the above embodiments may contain or consist of the amino acid sequence according to SEQ ID NO:149.

[0109] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CL domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:150. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a CL domain and comprises or constitutes the sequence according to SEQ ID NO:150.

[0110] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain (CL domain) containing one or more substitution mutations. In some embodiments, the one or more substitution mutations in the CL domain comprise or consist of Q124E, and the substitution mutation position is according to an EU number. In some embodiments, the one or more substitution mutations in the CL domain comprise or consist of R108A, T109S, or a combination thereof, and the substitution mutation position is according to an EU number. In some embodiments, the one or more substitution mutations in the CL domain comprise or consist of R108A, T109S, Q124E, or a combination thereof, and the substitution mutation position is according to an EU number.

[0111] In some embodiments, the CL domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:152. In some embodiments, the CL domain comprises or consists of the amino acid sequence according to SEQ ID NO:152.

[0112] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:153. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain and comprising or consisting of the sequence according to SEQ ID NO:153.

[0113] In some embodiments, the CL domain contains one or more substitution mutations. In some embodiments, the one or more substitution mutations in the CL domain contain E123K, Q124R, or a combination thereof, or consist of such mutations, and wherein the substitution mutation location is according to an EU number.

[0114] In some embodiments, the CL domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:155. In some embodiments, the CL domain comprises or consists of the amino acid sequence according to SEQ ID NO:155.

[0115] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:156. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain and comprising or consisting of the sequence according to SEQ ID NO:156.

[0116] In some implementations, one or more substitution mutations in the CL domain comprise E123R, Q124K, or a combination thereof, or consist of such mutations, and the substitution mutation location is determined according to the EU number.

[0117] In some embodiments, the CL domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:158. In some embodiments, the CL domain comprises or consists of the amino acid sequence according to SEQ ID NO:158.

[0118] In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain and having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:159. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence encoding a light chain constant domain and comprising or consisting of the sequence according to SEQ ID NO:159.

[0119] In some implementations, such as those provided herein, the polynucleotide encodes an immunoglobulin chain, wherein:

[0120] (i) The polynucleotide comprises a ribonucleic acid sequence encoding a CH2 domain, and the CH2 domain comprises one or more substitution mutations, wherein the one or more substitution mutations comprise G236A, A330L, I332E, or a combination thereof, or constitute thereof.

[0121] (ii) The polynucleotide comprises a ribonucleic acid sequence encoding a CH3 domain, and the CH3 domain comprises one or more substitution mutations, wherein the one or more substitution mutations comprise Y349C, S354C, T366S, T366W, L368A, Y407V, M428L, N434S, or combinations thereof, or constitute a subset thereof.

[0122] (iii) The polynucleotide comprises a ribonucleic acid sequence encoding a CH1 domain, and the CH1 domain comprises one or more substitution mutations, wherein the one or more substitution mutations comprise or consist of K147E, K213D, or a combination thereof.

[0123] (iv) The polynucleotide comprises a ribonucleic acid sequence encoding a CL domain, and the CL domain comprises one or more substitution mutations, wherein the one or more substitution mutations comprise R108A, T109S, E123K, E123R, Q124E, Q124K, Q124R, or combinations thereof, or constitute thereof.

[0124] (v) Their combination;

[0125] The substitution mutation sites are based on EU numbers.

[0126] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1307. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1307.

[0127] In some embodiments, the polynucleotides provided herein comprise a ribonucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the ribonucleic acid sequence according to SEQ ID NO:1306. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1306.

[0128] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1310. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1310.

[0129] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as the ribonucleic acid sequence according to SEQ ID NO:1309. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1309.

[0130] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1316. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1316.

[0131] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1315. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1315.

[0132] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1325. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1325.

[0133] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1324. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1324.

[0134] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1319. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1319.

[0135] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1318. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1318.

[0136] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1331. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1331.

[0137] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as the ribonucleic acid sequence according to SEQ ID NO:1330. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1330.

[0138] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1334. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1334.

[0139] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1333. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1333.

[0140] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1337. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1337.

[0141] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1336. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1336.

[0142] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1340. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1340.

[0143] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as the SEQ ID NO:1339. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1339.

[0144] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1313. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1313.

[0145] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1312. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1312.

[0146] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1328. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1328.

[0147] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1327. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1327.

[0148] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1355. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1355.

[0149] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1354. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1354.

[0150] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1322. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the amino acid sequence according to SEQ ID NO:1322.

[0151] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1321. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1321.

[0152] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1343. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the sequence according to SEQ ID NO:1343.

[0153] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1342. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1342.

[0154] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1346. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the sequence according to SEQ ID NO:1346.

[0155] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1345. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1345.

[0156] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1349. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the sequence according to SEQ ID NO:1349.

[0157] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1348. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1348.

[0158] In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1352. In some embodiments, the polynucleotides provided herein encode immunoglobulin chains comprising or consisting of the sequence according to SEQ ID NO:1362.

[0159] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO:1351. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO:1351.

[0160] In some embodiments, as provided herein, the polynucleotides encode immunoglobulin chains comprising amino acid sequences having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequences according to SEQ ID NO: 1358, 1361, 1364, 1367, 1370, 1373, 1376, 1379, 1382, 1385, 1388, 1391, 1394, 1397, 1400, 1403, 1406, 1409, 1412, 1415, 1418, 1421, 1424, 1427, 1430, 1433, 1436, 1439, 1442, 1445, 1448, 1451, 1454, 1457, or 1460. In some embodiments, as provided herein, the polynucleotides encode immunoglobulin chains comprising or composed of sequences according to SEQ ID NO: 1358, 1361, 1364, 1367, 1370, 1373, 1376, 1379, 1382, 1385, 1388, 1391, 1394, 1397, 1400, 1403, 1406, 1409, 1412, 1415, 1418, 1421, 1424, 1427, 1430, 1433, 1436, 1439, 1442, 1445, 1448, 1451, 1454, 1457, or 1460.

[0161] In some embodiments, the polynucleotides provided herein comprise at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as SEQ ID NO: 1357, 1360, 1363, 1366, 1369, 1372, 1375, 1378, 1381, 1384, 1387, 1390, 1393, 1396, 1399, 1402, 1405, 1408, 1411, 1414, 1417, 1420, 1423, 1426, 1429, 1432, 1435, 1438, 1441, 1444, 1447, 1450, 1453, 1456, or 1459. In some embodiments, the polynucleotides provided herein comprise or consist of the ribonucleic acid sequence according to SEQ ID NO: 1357, 1360, 1363, 1366, 1369, 1372, 1375, 1378, 1381, 1384, 1387, 1390, 1393, 1396, 1399, 1402, 1405, 1408, 1411, 1414, 1417, 1420, 1423, 1426, 1429, 1432, 1435, 1438, 1441, 1444, 1447, 1450, 1453, 1456, or 1459.

[0162] In some implementations, the polynucleotide contains a ribonucleic acid sequence encoding a secretion signal.

[0163] In some implementations, the secretion signal comprises a ribonucleic acid sequence according to SEQ ID NO:2, 4, 6 or 8.

[0164] In some implementations, the polynucleotide contains one or more non-coding sequence elements.

[0165] In some implementations, one or more non-coding sequence elements enhance RNA stability and / or translation efficiency.

[0166] In some implementations, one or more non-coding sequence elements comprise a 3' untranslated region (UTR), a 5' UTR, a 5'-cap, a polyadenine (polyA) tail, or a combination thereof.

[0167] In some implementations, the polyA tail is or includes a modified polyA sequence, preferably a discontinuous polyA tail.

[0168] In some implementations, the polyA tail contains or consists of a sequence that is at least 90%, at least 95%, or at least 99% identical to or constitutes the same as SEQ ID NO:416.

[0169] In some implementations, the 3'UTR contains or is composed of a nucleic acid sequence that is at least 90%, at least 95%, or at least 99% identical to or constitutes the same as SEQ ID NO:14.

[0170] In some implementations, the 5'UTR contains or is composed of a nucleic acid sequence that is at least 90%, at least 95%, or at least 99% identical to or constitutes the same as SEQ ID NO:10.

[0171] In some implementations, the 5'-cap is (m27,3'-O)Gppp(m2'-O)ApG.

[0172] In some embodiments, the polynucleotide comprises one or more modified ribonucleotides. In some embodiments, one or more modified ribonucleotides comprise pseudouridine.

[0173] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:850. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:850.

[0174] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:851. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:851.

[0175] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:853. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:853.

[0176] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:900. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:900.

[0177] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:901. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:901.

[0178] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:905. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:905.

[0179] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:906. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:906.

[0180] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:910. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:910.

[0181] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:950. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:950.

[0182] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:898. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:898.

[0183] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:947. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:947.

[0184] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:997. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:997.

[0185] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:1000. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1000.

[0186] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:1048. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1048.

[0187] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:1096. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1096.

[0188] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the ribonucleic acid sequence according to SEQ ID NO:1144. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1144.

[0189] In some embodiments, the polynucleotide comprises at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:850-1190. In some embodiments, the polynucleotide comprises or is composed of the ribonucleic acid sequence of any of SEQ ID NO:850-1190.

[0190] In some implementations, the polynucleotide is a non-natural polynucleotide.

[0191] In some implementations, the polynucleotide is an engineered polynucleotide.

[0192] In some implementations, the polynucleotide is an isolated polynucleotide.

[0193] In addition, this disclosure also provides compositions comprising one or more polynucleotides as described herein.

[0194] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1307; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1313.

[0195] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1306; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1312.

[0196] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1310; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1313.

[0197] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1309; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1312.

[0198] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1316; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1313.

[0199] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1315; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1312.

[0200] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1319; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1322.

[0201] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1318; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1321.

[0202] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1331; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1322.

[0203] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1330; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1321.

[0204] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1334; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1322.

[0205] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1333; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1321.

[0206] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1337; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1322.

[0207] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1336; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1321.

[0208] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1340; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1322.

[0209] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1339; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1321.

[0210] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1325; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1328.

[0211] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1324; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1327.

[0212] In some embodiments, the composition comprises or consists of the following: a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1325; and a polynucleotide encoding an immunoglobulin chain comprising an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid sequence according to SEQ ID NO:1355.

[0213] In some embodiments, the composition comprises or consists of the following: a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1324; and a polynucleotide comprising at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the same ribonucleic acid sequence as any of SEQ ID NO:1354.

[0214] In some embodiments, the composition further comprises lipid nanoparticles, polymeric complexes (PLX), lipotropic polymeric complexes (LPLX), or liposomes, wherein one or more polynucleotides are wholly or partially encapsulated within the lipid nanoparticles, polymeric complexes (PLX), lipotropic polymeric complexes (LPLX), or liposomes.

[0215] In some embodiments, the composition further comprises lipid nanoparticles, wherein one or more polynucleotides are encapsulated within the lipid nanoparticles.

[0216] In some implementations, lipid nanoparticles target hepatocytes.

[0217] In some implementations, lipid nanoparticles target secondary lymphoid organ cells.

[0218] In some implementations, lipid nanoparticles target lung cells.

[0219] In some implementations, the lipid nanoparticles are cationic lipid nanoparticles.

[0220] In some embodiments, the lipid nanoparticles each comprise polymer-conjugated lipids, cationic lipids, and one or more neutral lipids.

[0221] In some implementations, the polymer-conjugated lipid comprises a PEG-conjugated lipid.

[0222] In some embodiments, the polymer-conjugated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide.

[0223] In some embodiments, one or more neutral lipids comprise 1,2-distearate-sn-glycerol-3-phosphocholine (DPSC).

[0224] In some implementations, one or more neutral lipids include cholesterol.

[0225] In some embodiments, the cationic lipid comprises ((3-hydroxypropyl)azanidinediyl)bis(nonane-9,1-diyl)bis(2-butyloctanoate).

[0226] In some embodiments, the lipid nanoparticles each comprise 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide, DPSC, cholesterol, and bis(2-butyloctanoic acid)((3-hydroxypropyl)azanidinediyl)bis(nonane-9,1-diyl) ester.

[0227] In some embodiments, the lipid nanoparticles comprise about 1-2.5 mol% of polymer-conjugated lipids of total lipids, 35-65 mol% of cationic lipids of total lipids, and one or more neutral lipids present at 35-65 mol% of total lipids.

[0228] In some implementations, the lipid nanoparticles have an average diameter of about 50-150 nm.

[0229] This disclosure also provides pharmaceutical compositions. In some embodiments, the pharmaceutical composition comprises the composition provided herein and at least one pharmaceutically acceptable excipient.

[0230] In some embodiments, the drug comprises a cryoprotectant. In some embodiments, the drug comprises a buffered aqueous solution.

[0231] In addition, this disclosure also provides methods.

[0232] In some implementations, the method includes administering the pharmaceutical composition provided herein to a subject.

[0233] In some embodiments, the pharmaceutical compositions provided herein are used to treat HIV, including administering the pharmaceutical compositions to a subject.

[0234] In some embodiments, the pharmaceutical compositions provided herein are used for HIV prevention, including administering the pharmaceutical compositions to a subject.

[0235] In some embodiments, the methods or pharmaceutical compositions provided herein for use include administering the pharmaceutical composition to a subject, which results in the expression of an immunoglobulin chain of an antibody agent, an antibody agent, or both in the subject's body.

[0236] In some implementations, the immunoglobulin chain of the antibody agent, the antibody agent, or both are expressed in the subject's plasma or serum at a titer of at least 1 μg / ml.

[0237] In some implementations, when tested in a TZM-bl cell pseudovirus neutralization assay at antibody concentrations up to 25 μg / ml, the antibody showed a geometric mean IC50 of less than 0.3 μg / ml for five neutralizing strains in the global reference group.

[0238] In some implementations, when tested in a TZM-bl cell pseudovirus neutralization assay at antibody concentrations up to 25 μg / ml, the antibody is able to neutralize one or more HIV strains.

[0239] In some implementations, the antibody agent is capable of neutralizing one or more HIV strains, with the neutralization level being up to three times the level of an equivalent amount of recombinant baseline antibody.

[0240] In some implementations, the recombinant baseline antibody is an unmodified wild-type IgG antibody containing the same HCDR1, HCDR2, HCDR2, LCDR1, LCDR2, and LCDR3 as the antibody agent.

[0241] In some embodiments, administering the pharmaceutical composition to a subject includes administering one or more doses of the pharmaceutical composition to the subject. In some embodiments, one or more doses of the pharmaceutical composition are administered to the subject weekly. In some embodiments, one or more doses of the pharmaceutical composition are administered to the subject every two weeks.

[0242] In some embodiments, the drug composition is administered intravenously. In some embodiments, the drug composition is administered intramuscularly. In some embodiments, the drug composition is administered subcutaneously.

[0243] In some implementations, the subjects are HIV-infected or at risk of developing HSV infection.

[0244] In some implementations, the method is a treatment for HIV infection.

[0245] In some implementations, the method is a method for preventing HIV infection.

[0246] This article also provides for the uses of the polynucleotides, compositions and pharmaceutical compositions described herein.

[0247] In some embodiments, the use of the compositions or pharmaceutical compositions as provided herein for treating a subject with HIV is provided.

[0248] In some embodiments, the use of the compositions or pharmaceutical compositions as provided herein for the prevention of HIV in a subject is provided.

[0249] In some implementations, the subjects are HIV-infected or at risk of developing HSV infection.

[0250] Furthermore, this disclosure also provides methods for generating antibody agents. In some embodiments, the method includes administering a composition or pharmaceutical composition as provided herein to cells, causing the cells to express and secrete the antibody agent.

[0251] In some implementations, the cells are liver cells.

[0252] In some implementations, the cells are inside the subject's body.

[0253] In some implementations, the cells are isolated cells.

[0254] In some embodiments, the antibody agent is generated at a treatment-relevant plasma concentration or treatment-relevant serum concentration. In some embodiments, the treatment-relevant plasma concentration or treatment-relevant serum concentration is at least 1 μg / ml.

[0255] This disclosure also provides methods for determining one or more characteristics of an antibody agent expressed by the polynucleotide, composition, or pharmaceutical composition provided herein. In some embodiments, the polynucleotide, composition, or pharmaceutical composition provided herein is introduced into cells. In some embodiments, one or more characteristics include: (i) the protein expression level of the antibody agent; (ii) the binding specificity of the antibody agent to the Apex binding site of HIV; (iii) the efficacy of the antibody agent in mediating target cell death via antibody-dependent cytotoxicity (ADCC); and (iv) the efficacy of the antibody agent in mediating target cell death via complement-dependent cytotoxicity (CDC).

[0256] This disclosure also provides methods for contacting cells with the polynucleotides, compositions, or pharmaceutical compositions provided herein. In some embodiments, the method further includes detecting antibodies produced by the cells.

[0257] In some implementations, the cells are liver cells.

[0258] In some implementations, the determining step includes comparing one or more characteristics of the antibody agent with one or more characteristics of a reference antibody that specifically binds to the Apex binding site of HIV.

[0259] In some implementations, the determination step includes assessing that the protein expression level of the antibody agent is above a threshold level.

[0260] In some implementations, the threshold level is sufficient to induce ADCC.

[0261] In some implementations, the determination step includes assessing the binding of the antibody agent to the Apex (e.g., V1V2 region) binding site of HIV.

[0262] In some implementations, the determination step includes evaluating the antibody agent in a TZM-bl cell pseudovirus neutralization assay at antibody agent concentrations up to 25 μg / ml.

[0263] In some implementations, the cells are present within the subject's body.

[0264] In some implementations, the cells are isolated cells.

[0265] In some implementations, one or more features include antibody levels in one or more tissues of the subject.

[0266] This disclosure also provides manufacturing methods. In some embodiments, the method includes (A) determining one or more characteristics of the polynucleotide, composition, or pharmaceutical composition provided herein, said one or more characteristics comprising or consisting of:

[0267] (i) The length and / or sequence of the polynucleotide;

[0268] (ii) Integrity of polynucleotides;

[0269] (iii) The presence and / or location of one or more chemical moieties of a polynucleotide;

[0270] (iv) The level of antibody expression when polynucleotides are introduced into cells;

[0271] (v) Stability of polynucleotides or combinations thereof;

[0272] (vi) The level of antibody in biological samples from organisms in which polynucleotides have been introduced;

[0273] (vii) The binding specificity of the antibody agent expressed by the polynucleotide, optionally binding to the CD4 binding site of HIV;

[0274] (viii) The efficacy of antibody agents in mediating target cell death through ADCC;

[0275] (ix) The efficacy of antibody agents in mediating target cell death through complement-dependent cytotoxicity (CDC);

[0276] (x) Lipid identity and amount / concentration in the composition;

[0277] (xi) The size of the lipid nanoparticles in the composition;

[0278] (xii) The polydispersity of lipid nanoparticles in the composition;

[0279] (xiii) The amount / concentration of polynucleotides in the composition;

[0280] (xiv) The degree of encapsulation of polynucleotides within lipid nanoparticles;

[0281] (xv) levels of double-stranded RNA; and

[0282] (xvi) Their combinations;

[0283] (B) Compare one or more characteristics of the polynucleotide with one or more characteristics of an appropriate reference standard; and

[0284] (C)(i) If the comparison indicates that the polynucleotide or its composition meets or exceeds the reference standard, then specify one or more other steps for the manufacture and / or dispensing of the polynucleotide or its composition; or

[0285] (C)(ii) If the comparison indicates that the polynucleotide or its composition does not meet or exceeds the reference standard, alternative action shall be taken.

[0286] In some implementations, the polynucleotide is evaluated, and one or more other steps of step (C)(i) are at least or include the formulation of a polynucleotide.

[0287] In some embodiments, the composition or pharmaceutical composition is evaluated, and one or more other steps of step (C)(i) are or include the release and dispensing of the composition or pharmaceutical composition.

[0288] Therefore, this disclosure provides a technique that allows multiple anti-HIV antibodies to be expressed in a subject, thereby increasing the breadth and potency of anti-HIV antibodies coexisting in the subject and reducing the possibility of viral escape.

[0289] This document describes the provided techniques in more detail, including exemplary polynucleotides, compositions containing such polynucleotides, and methods for manufacturing and using such polynucleotides. Attached Figure Description

[0290] Figure 1A schematic diagram showing the structure of the HIV genome (Figure A) and HIV viral particles (Figure B) is presented. The figures are modified from Musumeci et al., 2015, Molecules (which is incorporated herein by reference in its entirety).

[0291] Figure 2 This figure illustrates potential epitope regions on HIV viral particles that antibody agents (e.g., broadly neutralizing antibodies (bNAb) or their variants) can bind to. The figures are modified from McCoy and Burton, 2017 Immunol Rev. (which is incorporated herein by reference in its entirety).

[0292] Figure 3 An exemplary therapeutic strategy for delivering and expressing anti-HIV RibobNab using RiboMab technology as described herein is illustrated.

[0293] Figure 4 Exemplary forms of PGDM1400 RibobNab as described herein are shown. Exemplary forms may include IgG (Figure A), CrossMab, etc. CH1-CLx (Figure B), CrossMab CH1-CLv (Figure D) or various orientations / connectors of scFv-Fc RibobNAb (Figures C and E).

[0294] Figure 5 An exemplary Fc modification of the PGDM1400 RibobNab as described herein is shown. The exemplary RiboMab form may include an unmodified Fc domain (Figure A), or modifications as shown in Figures BD, including GAALIE / GAIE / GA / IE (Figure B), L / S (Figure D), and / or mortar RibobNAb (Figure C).

[0295] Figure 6 A schematic diagram of exemplary polynucleotides encoding the heavy chain (Figure A) and light chain (Figure B) of an exemplary PGDM1400 IgG1 antibody agent is shown.

[0296] Figure 7 The spectrum of broad-spectrum HIV-neutralizing antibodies (including PGDM1400) is shown in tests against a group of 109 pseudoviruses.

[0297] Figure 8 A schematic diagram of exemplary polynucleotides encoding the scFv-Fc PGDM1400 antibody agent is shown: PGDM1400 scFV-Fc VH-LL4 / 5-VL (Figure A) and PGDM1400scFV-Fc VL-LL4 / 5-VH (Figure B).

[0298] Figure 9 Example encoding of PGDM1400 CrossMab is shown. CH1-CLx A schematic diagram of exemplary polynucleotides of the heavy chain (Figure A) and light chain (Figure B) of an antibody agent.

[0299] Figure 10 Example encoding of PGDM1400 CrossMab is shown. CH1-CLcv A schematic diagram of exemplary polynucleotides of the heavy chain (Figure A) and light chain (Figure B) of an antibody agent.

[0300] Figure 11 Exemplary concentrations of PGDM1400 and PGDM1400 L / S RibobNAb, as determined by the Gyros ELISA in Example 5, compared to the control RiboMab, are shown.

[0301] Figure 12 Exemplary Western blot analyses of PGDM1400 and PGDM1400 L / S RibobNAb compared to control RiboMab are shown under non-reducing conditions in Example 5.

[0302] Figure 13 Exemplary concentrations of scFv-Fc PGDM1400 L / S RibobNAb (e.g., VH-LL4-VL, VL-LL5-VH, VH-LL5-VL, and VL-LL4-VH) compared to the IgG control RiboMab and parental IgG, as determined by the Gyros ELISA in Example 6, are shown.

[0303] Figure 14 Exemplary Western blot analysis of scFv-FcPGDM1400 L / S RibobNAb (e.g., VH-LL4-VL, VL-LL5-VH, VH-LL5-VL, and VL-LL4-VH) under non-reducing conditions as shown in Example 6, compared to control RiboMab and parental IgG.

[0304] Figure 15 As shown by the Gyros ELISA in Example 7, compared with control RiboMab and parental Ab, CrossMab PGDM1400 L / S RibobNAb (e.g., CrossMab) CH1-CLcv and CrossMab CH1-CLx Example concentrations of ).

[0305] Figure 16As shown in Example 7, compared with control RiboMab and parental Ab, CrossMab PGDM1400 L / SRibobNAb (e.g., CrossMab) CH1-CLcv and Cross Mab CH1-CLx Exemplary protein blot analysis.

[0306] Figure 17 Exemplary pharmacokinetic (PK) profiles of PGDM1400 IgG and PGDM1400 IgG L / S RibobNAb compared to control RiboMab are shown in NSG mice (Fig. A) and NSG Tg32 mice (Fig. B), as determined by the Gyros ELISA in Example 8. PGDM1400RibobNAb was quantified using serum obtained from NSG mice and hFcRn NSG Tg32 mice. In vivo concentrations (μg / mL) are displayed on a logarithmic scale on the y-axis. The x-axis shows the time (days) of each blood sample.

[0307] Figure 18 An exemplary pharmacokinetic (PK) profile of PGDM1400 IgG, IgG L / S, and scFv-Fc L / S RibobNAb is shown. PGDM1400 RibobNAb was quantified using serum obtained from hFcRn NSG Tg32 mice. In vivo concentrations (μg / mL) are displayed on a logarithmic scale on the y-axis. The x-axis shows the time (days) of each blood sample. Results for PGDM1400VL-LL5-VH L / S scFv-Fc, as determined by the Gyros ELISA as described in Example 12, are depicted. Results for PGDM1400VL-LL5-VH L / S scFv-Fc, compared to PGDM1400 IgG and PGDM1400 L / S IgG, were analyzed at two different doses (30 μg and 19.56 μg) of PGDM1400 VL-LL5-VH L / S scFv-Fc and PGDM1400 L / S IgG.

[0308] Figure 19Exemplary results of the pseudovirus neutralization test (pVNT) are shown, in which TZM.b1 cells were exposed to PGDM1400scFv-Fc L / S (VH-LL4-VL, VL-LL5-VH, VH-LL5-VL, and VL-LL4-VH configurations) and pseudoviruses ZM53M.PB12, Du156.12, Q769.d22, 0330.v4.c3, R2184.c04, and 89-F1_2_25. Murine leukemia virus (MuLV) pseudovirus was used as a negative control. Results are expressed as IC50 and IC80 values ​​or antibody / IgG concentrations, with a 50% and 80% reduction in relative luminescent units (RLU) compared to untreated virus control wells.

[0309] Figure 20 Exemplary results for pVNT are shown, in which TZM.b1 cells were exposed to PGDM1400 scFv-Fc L / S (VH-LL4-VL, VH-LL5-VL, VL-LL4-VH, and VL-LL5-VH configurations) and pseudoviruses CAP304_2_00_F6_6, Ce1176_A3, 6980.v0.c31, 1012_11_TC21_3257, and PVO.4. Murine leukemia virus (MuLV) pseudovirus was used as a negative control. Results are expressed as IC50 and IC80 values ​​or antibody / IgG concentrations, showing a 50% and 80% reduction in relative luminescent units (RLU) compared to untreated virus control wells.

[0310] Figure 21 Exemplary results for pVNT are shown, in which TZM.b1 cells are exposed to PGDM1400 CrossMab. CH1-CLx L / S and PGDM1400 CrossMab CH1-CHcv L / SRibobNAb and pseudoviruses ZM53M.PB12, Du156.12, Q769.d22, 0330.v4.c3, R2184.c04, and 89-F1_2_25 were used. Murine leukemia virus (MuLV) pseudoviruses were used as negative controls. Results are expressed as IC50 and IC80 values ​​or antibody / IgG concentrations, showing a 50% and 80% reduction in relative luminescent units (RLU) compared to the untreated virus control wells.

[0311] Figure 22Exemplary results for pVNT are shown, in which TZM.b1 cells were exposed to PGDM1400 scFv-Fc(VL-LL5-VH)L / S RibobNAb and pseudoviruses CAP45, X2088_c9, CNE8, T250-4, QH0692.42, and 3103.v3.c10 (Fig. A) and C1080_c3, T278-50, ZM109F.PB4, and Du156.12 (Fig. B). Murine leukemia virus (MuLV) pseudovirus was used as a negative control. Results are expressed as IC50 and IC80 values ​​or antibody / IgG concentrations, with a 50% and 80% reduction in relative luminescent units (RLU) compared to untreated virus control wells.

[0312] definition

[0313] The compounds disclosed herein include those generally described above, and are further illustrated by the categories, subclasses, and species disclosed herein. Unless otherwise stated, the following definitions shall apply as used herein. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. Furthermore, the general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999 and “March's Advanced Organic Chemistry”, 5th edition, edited by Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0314] Unless otherwise stated, the structures described herein are intended to include all stereoisomers (e.g., enantiomers or diastereomers) of the structure, as well as all geometric or conformational isomers of the structure. For example, the R and S configurations of each stereocenter are considered part of this disclosure. Therefore, single stereochemical isomers of the provided compounds, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are within the scope of this disclosure. For example, in some cases, the provided compounds show one or more stereoisomers of the compound and, unless otherwise stated, represent each stereoisomer individually and / or as a mixture. Unless otherwise stated, all tautomer forms of the provided compounds are within the scope of this disclosure.

[0315] Unless otherwise stated, the structures described herein are intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures disclosed herein (including hydrogen replaced by deuterium or tritium, or carbon replaced by 13C-enriched carbon or 14C-enriched carbon) are within the scope of this disclosure.

[0316] About: When used herein to refer to a value, the term “about” means a value similar to the value referred to in the context. Generally, those skilled in the art will understand the extent of relevant variation covered by “about” in that context. For example, in some embodiments, the term “about” may cover a range of values ​​up to 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the value referred to.

[0317] Agent: As used herein, the term "agent" can refer to a physical entity. In some embodiments, an agent can be characterized by specific characteristics and / or effects. For example, as used herein, the term "therapeutic agent" refers to a physical entity that has a therapeutic effect and / or causes the desired biological and / or pharmacological effect. In some embodiments, an agent can be a compound, molecule, or entity of any chemical class, including, for example, small molecules, peptides, nucleic acids, sugars, lipids, metals, or combinations or complexes thereof.

[0318] Aliphatic: The term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is fully saturated or contains one or more unsaturated units, or a monocyclic or bicyclic hydrocarbon (also referred to herein as "alicyclic") that is fully saturated or contains one or more unsaturated units but is not aromatic, and has a single or more connection points with the rest of the molecule. Unless otherwise stated, the aliphatic group contains 1-12 aliphatic carbon atoms. In some embodiments, the aliphatic group contains 1-6 aliphatic carbon atoms (e.g., C64 ... 1-6 In some embodiments, the aliphatic group contains 1-5 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-5 In other embodiments, the aliphatic group contains 1-4 aliphatic carbon atoms (e.g., C46, ​​C56, C6 ... 1-4 In other embodiments, the aliphatic group contains 1-3 aliphatic carbon atoms (e.g., C360, C46, ​​C56, C6 ... 1-3 In other embodiments, the aliphatic group contains 1-2 aliphatic carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-2 Suitable aliphatic groups include, but are not limited to, straight-chain or branched-chain, substituted or unsubstituted alkyl, alkenyl or ynyl groups, and their hybrids. Preferred aliphatic groups are C1. 1-6 alkyl.

[0319] Alkyl: The term "alkyl" as used alone or as part of a larger part refers to having 1-12, 1-10, 1-8, 1-6, 1-4, 1-3 or 1-2 carbon atoms (e.g., C12, C23, C14, C23, C2 ... 1-12 C 1-10 C 1-8 C 1-6 C 1-4 C 1-3 Or C 1-2 The alkyl group is a saturated, optionally substituted straight-chain or branched hydrocarbon group. Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.

[0320] Alkylene: The term "alkylene" refers to a divalent alkyl group. In some embodiments, "alkylene" is a divalent straight-chain or branched alkyl group. In some embodiments, the "alkylene chain" is polymethylene, i.e., -(CH2). n - where n is a positive integer, such as 1 to 6, 1 to 4, 1 to 3, 1 to 2, or 2 to 3. The optionally substituted alkylene chain is polymethylene, wherein one or more methylene hydrogen atoms are optionally replaced by substituents. Suitable substituents include those described below with respect to substituted aliphatic groups, and also those described in this specification. It should be understood that two substituents of an alkylene group can together form a ring system. In some embodiments, two substituents can combine to form a 3- to 7-membered ring. Substituents can be on the same or different atoms. The suffix "-ene" or "-enyl" when attached to certain groups herein is intended to refer to the bifunctional portion of said group. For example, "-ene" or "-enyl" when attached to "cyclopropyl" becomes "cyclopropene" or "cyclopropenyl" and is intended to refer to a bifunctional cyclopropyl group, such as...

[0321] Alkenyl: The term "alkenyl" used alone or as part of a larger part refers to a group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-12 C 2-10 C 2-8 C 2-6 C 2-4 Or C 2-3 The optional substituted straight-chain, branched-chain, or cyclic hydrocarbon group of the alkenyl group. Exemplary alkenyl groups include vinyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or polycyclic system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.

[0322] Alkynyl: The term "alkynyl" used alone or as part of a larger part refers to a group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C4, C5, C6, C7, C8, C9 ... 2-12 C 2-10 C 2-8 C 2-6 C 2-4 Or C 2-3 The alkynyl group may be a straight-chain or branched hydrocarbon group that has been optionally substituted. Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.

[0323] Amino acid: In the broadest sense, as used herein, the term "amino acid" refers to a compound and / or substance that can, is, or has been incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, an amino acid has the general structure H₂N–C(H)(R)–COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. In some embodiments, the amino acids in the polypeptide (including carboxyl and / or amino-terminal amino acids) may contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified compared to the general structure by methylation, amidation, acetylation, polyethylene glycolation, glycosylation, phosphorylation, and / or substitution (e.g., substitution of amino, carboxylic acid groups, one or more protons, and / or hydroxyl groups). In some embodiments, such modification may, for example, alter the cycling half-life of a peptide containing the modified amino acid compared to a peptide containing the same unmodified amino acid in other respects. In some embodiments, such modification does not significantly alter the relevant activity of a peptide containing the modified amino acid compared to a peptide containing the same unmodified amino acid in other respects. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to the amino acid residues of a peptide.

[0324] Antibody Agent: As used herein, the term "antibody agent" refers to any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding to a particular antigen. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more humanized, primate-like, chimeric, or other sequence elements, as known in the art. In some embodiments, the term "antibody agent" is used to refer to one or more constructs or forms known or developed in the art for utilizing antibody structural and functional features in alternative presentations. For example, in some embodiments, antibody agents used according to this disclosure are selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., (etc.); CrossMab (e.g., CrossMab) CH1-CL CrossMab CH1-CLcv CrossMab with bispecificity of pestle and mortar CH1-CL Antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs) or sets thereof; single-chain Fv (scFv); scFv-Fc fusions; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel-shaped antibodies; masked antibodies (e.g., Small modular immunotherapies (“SMIPs™”); single-chain or tandem bifunctional antibodies VHH; Small antibodies; Ankylosing spondylogenetics or DART; TCR-like antibodies; MicroProteins; as well as In some embodiments, such antibodies and fragments of chains and / or fragments may be used in combination, for example, the scFv-Fc arm may be used in combination with a conventional antibody arm. In some embodiments, the antibody agent is a broad-spectrum neutralizing antibody agent (e.g., a broad-spectrum neutralizing antibody (bNab)). A “broad-spectrum neutralizing antibody agent” is an antibody agent capable of neutralizing two or more genetic variants (e.g., strains) of a virus (e.g., HIV). In some embodiments, the antibody may lack the covalent modifications (e.g., glycan linkages) that it would have in naturally occurring cases. In some embodiments, the antibody may contain linkages to covalent modifications (e.g., glycans, payloads (e.g., detectable portions, therapeutic portions, catalytic portions, etc.) or other side groups (e.g., polyethylene glycol, etc.). In many embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements identified by those skilled in the art as complementarity-determining regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR), said at least one CDR being substantially identical to a CDR found in a reference antibody. In some embodiments, the included CDR is substantially identical to a reference CDR because it is sequence-identical to the reference CDR, or contains between 1 and 5 amino acid substitutions. In some embodiments, the included CDR is substantially identical to a reference CDR because it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because it exhibits at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR because, compared to the reference CDR, the included CDR has at least one amino acid deleted, added, or substituted, but the included CDR has the same amino acid sequence as the reference CDR in all other respects. In some embodiments, the included CDR is substantially identical to the reference CDR because, compared to the reference CDR, the included CDR has 1-5 amino acids deleted, added, or substituted, but the included CDR has the same amino acid sequence as the reference CDR in all other respects. In some embodiments, the included CDR is substantially identical to the reference CDR because, compared to the reference CDR, the included CDR has at least one amino acid substituted, but the included CDR has the same amino acid sequence as the reference CDR in all other respects.In some embodiments, the included CDR is substantially identical to the reference CDR because, compared to the reference CDR, the included CDR has 1-5 amino acids missing, added, or substituted, but has the same amino acid sequence as the reference CDR in all other respects. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, the antibody agent is a polypeptide protein having a binding domain homologous to or substantially homologous to an immunoglobulin binding domain.

[0325] Aryl: The term "aryl" refers to a monocyclic and bicyclic system having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and each ring in the system contains three to seven ring members. In some embodiments, "aryl" contains between six and twelve total ring members (e.g., C6-C12). The term "aryl" may be used interchangeably with the term "aryl ring." In some embodiments, "aryl" refers to an aromatic ring system that may carry one or more substituents, including but not limited to phenyl, biphenyl, naphthyl, anthracene, etc. Unless otherwise stated, "aryl" is a hydrocarbon. In some embodiments, the "aryl" ring system is an aromatic ring (e.g., phenyl) fused with a non-aromatic ring (e.g., cycloalkyl). Examples of fused aryl rings include...

[0326] Related: When used herein, two events or entities are “related” to each other if the presence, level, extent, type, and / or form of one event or entity is related to the presence, level, extent, type, and / or form of another event or entity. For example, an entity is considered related to a particular disease, condition, or disorder if the presence, level, and / or form of a particular entity (e.g., polypeptide, genetic signature, metabolite, microorganism, etc.) is related to the incidence, susceptibility, severity, stage, etc., of that particular disease, condition, or disorder (e.g., within a relevant population). In some embodiments, two or more entities are physically “associated” to each other if they interact directly or indirectly such that they are physically close to each other and / or remain physically close. In some embodiments, two or more physically associated entities are covalently linked to each other; in some embodiments, two or more physically associated entities are not covalently linked to each other but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic forces, and combinations thereof.

[0327] Co-administration: As used herein, the term "co-administration" refers to the use of a composition described herein (e.g., a pharmaceutical composition) and one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents comprise at least one polynucleotide encoding another antibody agent (e.g., an anti-HIV antigen antibody agent). The combination of the composition described herein (e.g., a pharmaceutical composition) and the additional therapeutic agent may be used simultaneously or separately (e.g., sequentially in any order). In some embodiments, the composition described herein (e.g., a pharmaceutical composition) and the additional therapeutic agent may be combined in a pharmaceutically acceptable excipient, or they may be placed in separate excipients and delivered to target cells or administered to a subject at different times. Each of these cases is considered to fall within the meaning of "co-administration" or "combination" as long as the composition described herein (e.g., a pharmaceutical composition) and the additional therapeutic agent are delivered or administered sufficiently close in time such that the biological effects of each on the target cells or the treated subject have at least some temporal overlap.

[0328] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents (e.g., two or more antibody agents)). In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "dose" of the first regimen followed by any dose of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, administration of combination therapy may involve administering one or more agents or methods to a subject who is receiving other agents or methods in the combination. For clarity, combination therapy does not require the individual agents to be administered together in a single composition (or even necessarily simultaneously), but in some embodiments, two or more agents or their active portions may be administered together in a combination composition. In some embodiments, combination therapy comprises a polynucleotide encoding two or more antibody agents (e.g., an anti-HIV antibody agent).

[0329] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, conditions, etc., that may not be identical to each other, but are similar enough to allow comparisons between them, such that those skilled in the art will understand that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, a set of comparable conditions, situations, individuals, or groups is characterized by a number of substantially identical features and one or a few varying features. Those skilled in the art will understand, in any given context, what degree of identity is required between two or more such sets of agents, entities, situations, conditions, etc., to be considered comparable. For example, those skilled in the art will understand that a set of situations, individuals, or groups is comparable to each other when it is characterized by a sufficient number and type of substantially identical features to ensure that the difference in results or observed phenomena obtained under different sets of situations, individuals, or groups, or with different sets of situations, individuals, or groups, is caused by or indicates a change in those varying features.

[0330] Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” can be used to indicate the position / characteristic of a structural element in a compound or composition relative to another compound or composition (e.g., relative to a suitable reference compound or composition). For example, in some embodiments, monomeric residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as “corresponding to” residues in a suitable reference polymer. For example, those skilled in the art will appreciate that, for simplicity, residues in polypeptides are typically named using a canonical numbering system based on a reference related polypeptide, so the amino acid “corresponding to” the residue at position 190, for example, does not actually have to be the 190th amino acid in a particular amino acid chain, but rather corresponds to the 190th visible residue in the reference polypeptide; those skilled in the art can readily understand how to identify the “corresponding” amino acid. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, which can be used, for example, to identify “corresponding” residues in peptides and / or nucleic acids according to this disclosure. Those skilled in the art will also understand that, in some cases, the term “corresponding” can be used to describe an event or entity that shares similarity with another event or entity (e.g., a suitable reference event or entity). To give just one example, a gene or protein in one organism can be described as “corresponding” to a gene or protein from another organism, in order to indicate in some embodiments that it plays a similar role or performs a similar function and / or that it exhibits a particular degree of sequence identity or homology, or shares specific characteristic sequence elements.

[0331] Cycloaliphatic: As used herein, the term "cycloaliphatic" refers to a monocyclic C14 molecule that is fully saturated or contains one or more unsaturated units but is not aromatic, and has a single or more connection points with the rest of the molecule. 3-8 Hydrocarbons or bicyclic C 6-10 hydrocarbon.

[0332] Cycloalkyl: As used herein, the term "cycloalkyl" refers to a saturated cyclic monocyclic or polycyclic system with optional substitution of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0333] Source: In the context of "derived from" an amino acid sequence (peptide or polypeptide) of a specified amino acid sequence (peptide or polypeptide), it refers to a structural analog of the specified amino acid sequence. In some embodiments, the amino acid sequence derived from a particular amino acid sequence has the same, substantially the same, or homologous amino acid sequence as the specific sequence or a fragment thereof. The amino acid sequence derived from a particular amino acid sequence may be a variant of the specific sequence or a fragment thereof. For example, antibody agents used according to this disclosure may include amino acid sequences (e.g., CDRs, variable domains, constant domains, etc.) derived from other antibodies (e.g., naturally occurring antibodies).

[0334] Detection: The term “detection” is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest in a sample or any form of measurement of the entity of interest. Thus, “detection” can include determining, measuring, evaluating, or determining the presence or absence, level, quantity, and / or location of an entity of interest. This includes both quantitative and qualitative determinations, measurements, or evaluations, including semi-quantitative ones. Such determinations, measurements, or evaluations can be relative (e.g., when an entity of interest is detected relative to a control reference) or absolute. Therefore, the term “quantitative” in the context of quantifying an entity of interest can refer to absolute or relative quantification. Absolute quantification can be achieved by relating the level of a detected entity of interest to a known control standard (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the detected levels or quantities between two or more distinct entities of interest to provide a relative quantification of each of the two or more distinct entities of interest (i.e., relative to each other).

[0335] Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" (or "treatment regimen") can be used to refer to a set of unit doses (usually more than one) that are typically administered individually to a subject at intervals. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses.

[0336] Encoding: As used herein, the term "encode" refers to the sequence information of a first molecule that directs the production of a second molecule having a defined nucleotide sequence (e.g., a polynucleotide) or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., through transcription involving a DNA-dependent RNA polymerase). An RNA molecule can encode a polypeptide (e.g., through translation). Thus, if transcription and translation of RNA corresponding to a gene produces a polypeptide in a cell or other biological system, then the gene, cDNA, or RNA molecule encodes the polypeptide. In some embodiments, the coding region of a polynucleotide encoding a target antigen refers to the coding strand whose nucleotide sequence is identical to the polynucleotide sequence of such a target antigen. In some embodiments, the coding region of a polynucleotide encoding a target antigen refers to the non-coding strand of such a target antigen, which can be used as a transcription template for a gene or cDNA.

[0337] Engineered: Generally, the term “engineered” refers to aspects that have been artificially manipulated. For example, a polynucleotide is considered “engineered” when two or more sequences that are not linked together in their natural order are directly linked to each other through artificial manipulation, and / or when a particular residue in a polynucleotide is not naturally present and / or is linked to an entity or part of it that is not naturally linked, through artificial means.

[0338] Epitope: As used herein, the term "epitaph" refers to a portion that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope can be recognized by T cells, B cells, or antibodies. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen takes an associated three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen takes such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically spaced apart when the antigen takes an alternative conformation (e.g., is linearized). Therefore, in some embodiments, an epitope of an antigen may include continuous or discontinuous portions of the antigen. In some embodiments, an epitope is or comprises a T-cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, about 10 to about 25 amino acids, about 5 to about 15 amino acids, about 5 to 12 amino acids, or about 6 to about 9 amino acids.

[0339] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the production of a gene product from the nucleic acid sequence. In some embodiments, the gene product may be a transcript, such as the polynucleotides provided herein. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence involves one or more of the following: (1) the production of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) the translation of RNA into a polypeptide or protein; and / or (4) post-translational modifications of the polypeptide or protein.

[0340] Heteroaliphatic: As used herein, the term "heteroaliphatic" or "heteroaliphatic group" refers to a hydrocarbon moiety with optional substitutions of one to five heteroatoms in addition to carbon atoms. It can be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic") and can be fully saturated, or may contain one or more unsaturated units but is not aromatic. The term "heteroatom" means nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur and any quaternized form of basic nitrogen. The term "nitrogen" also includes substituted nitrogen. Unless otherwise stated, a heteroaliphatic group contains 1 to 10 carbon atoms, wherein 1 to 3 carbon atoms are optionally and independently substituted with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, the heteroaliphatic group contains 1 to 4 carbon atoms, wherein 1 to 2 carbon atoms are optionally and independently substituted with heteroatoms selected from oxygen, nitrogen, and sulfur. In other embodiments, the heteroaliphatic group contains 1 to 3 carbon atoms, wherein 1 carbon atom is optionally and independently substituted with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, straight-chain or branched heteroalkyl, heteroalkenyl, and heteroynyl groups. For example, heteroaliphatic groups of 1 to 10 atoms include the following exemplary groups: O-CH3, -CH2-O-CH3, -O-CH2-CH2-O-CH2-CH2-O-CH3, etc.

[0341] Heteroaryl: The terms “heteroaryl” and “hetero-aryl-”, used alone or as part of a larger portion (e.g., “heteroarylalkyl” or “heteroarylalkoxy”), refer to a monocyclic or bicyclic group having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 π electrons shared in a cyclic array; and having one to five heteroatoms in addition to carbon atoms. Heteroaryl groups include, but are not limited to, thiophene, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiazolyl, thiazolyl, pyridinyl, pyrazinyl, indazinyl, purine, naphthinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridinyl, imidazo[4,5-b]pyridinyl, imidazo[4,5-c]pyridinyl, pyrrolopyridinyl, pyrrolopyrazinyl, thiophenolopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl. As used herein, the terms “heteroaryl” and “heteroary-” also include groups in which a heteroaryl ring is fused with one or more aryl, cycloaliphatic or heterocyclic rings, wherein the linking group or linking point is on the heteroaryl ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Non-limiting examples include indolyl, isoindolyl, benzothiophenyl, benzofuranyl, dibenzofuranyl, inzolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzooxazolyl, quinolinyl, isoquinolinyl, terpineyl, phthalazinyl, quinazolinyl, quinoxolinyl, 4H-quinazinyl, carbazoleyl, acridineyl, phenazinyl, phenothiazinyl, phenotoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-1,4-oxazin-3(4H)-one, 4H-thieno[3,2-b]pyrrole, and benzoisooxazolyl. The term "heteroaryl" may be used interchangeably with the terms "heteroary ring," "heteroaryl," or "heteroary aromatic," any of which includes an optionally substituted ring.

[0342] Heteroatoms: As used herein, the term “heteroatoms” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, as well as any quaternized form of basic nitrogen.

[0343] Heterocycle: As used herein, the terms “heterocycle,” “heterocycl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, 6- to 10-membered bicyclic, or 10- to 16-membered polycyclic heterocyclic moiety that is saturated or partially unsaturated and has one or more (such as one to four) heteroatoms as defined above, in addition to a carbon atom. When used to refer to the ring atom of a heterocycle, the term “nitrogen” includes substituted nitrogen. For example, in a saturated or partially unsaturated ring having 0 to 3 heteroatoms selected from oxygen, sulfur, or nitrogen, nitrogen can be N (as in 3,4-dihydro-2H-pyrrole), NH (as in pyrrolidinyl), or NR. + (e.g., in N-substituted pyrrolidinyl groups). The heterocycle can be attached to its side group at any heteroatom or carbon atom to produce a stable structure, and any ring atom can optionally be substituted. Examples of such saturated or partially unsaturated heterocyclic groups include, but are not limited to, azeti dinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazineyl, dioxalyl, dioxopentyl, diazaphenyl, oxazphenyl, thioazphenyl, morpholinyl, and thiomorpholinyl. The heterocyclic group can be monocyclic, bicyclic, tricyclic, or polycyclic, preferably monocyclic, bicyclic, or tricyclic, more preferably monocyclic or bicyclic. Bicyclic heterocycles also include groups in which the heterocycle is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indololinyl, isoindolinyl, benzodioxanepentenyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. Bicyclic heterocycles can also be spirocyclic systems (e.g., 7- to 11-membered spirocyclic fused heterocycles, wherein the heterocycle has one or more heteroatoms as defined above (e.g., one, two, three, or four heteroatoms) in addition to a carbon atom). Bicyclic heterocycles can also be bridged ring systems (e.g., 7- to 11-membered bridged heterocycles having one, two, or three bridging atoms).

[0344] Homology: As used herein, the term "homology" or "homogeneity" refers to the overall relevance between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered "homological" of each other if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA and / or RNA molecules) and / or polypeptide molecules are considered “homologous” to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing chemically related residues at corresponding positions). For example, as is well known to those skilled in the art, certain amino acids are generally classified as “hydrophobic” or “hydrophilic” amino acids that are similar to each other, and / or have “polar” or “nonpolar” side chains. Substituting one amino acid for another of the same type is generally considered a “homologous” substitution.

[0345] Identity: As used herein, the term "identity" refers to the overall relevance between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered "substantially identical" to each other if their sequences are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. The percentage of identity between two nucleic acid or polypeptide sequences can be calculated, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). In some implementations, the length of the sequence compared for comparative purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of the reference sequence. Nucleotides at the corresponding positions are then compared. The molecule is considered identical at that position when a position in the first sequence is occupied by the same residue (e.g., a nucleotide or amino acid) as the corresponding position in the second sequence. The percentage of identity between the two sequences varies with the number of shared positions, where the length needs to be introduced to achieve optimal alignment of the two sequences, taking into account the number of gaps and the length of each gap. Mathematical algorithms can be used to perform the comparison of sequences and the determination of the percentage of identity between two sequences. For example, the algorithm of Meyers and Miller, 1989, incorporated into the ALIGN program (version 2.0), can be used to determine the percentage of identity between two nucleotide sequences. In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program utilize a PAM120 weighted residue table, a nick length penalty of 12, and a nick penalty of 4. Alternatively, the GAP program in the GCG software package can be used to determine the percentage of identity between two nucleotide sequences using the NWSgapdna.CMP matrix.

[0346] Increased, induced, or decreased: As used herein, these terms, or grammatically equivalent comparative terms, indicate values ​​relative to an equivalent reference measurement. For example, in some embodiments, an assessment value obtained with the provided composition (e.g., a pharmaceutical composition) may be “increased” relative to an assessment value obtained with an equivalent reference composition. Or, additionally, in some embodiments, an assessment value obtained in a subject may be “increased” relative to an assessment value obtained in the same subject under different conditions (e.g., before or after an event; or with or without an event, such as administration of a composition as described herein (e.g., a pharmaceutical composition)) or in different equivalent subjects (e.g., in equivalent subjects different from the subject of interest, who were previously exposed to conditions, for example, without administration of a composition as described herein (e.g., a pharmaceutical composition)). In some embodiments, comparative terms refer to statistically relevant differences (e.g., universality and / or magnitude sufficient to be statistically relevant). Those skilled in the art will appreciate, or will be able to readily determine, that in a given context... In this context, the degree and / or prevalence of the difference required or sufficient to achieve such statistical significance refers to the level of difference. In some embodiments, the term "reduced" or its equivalent means a reduction of at least 5%, at least 10%, at least 20%, at least 50%, at least 75%, or higher in the level of the assessed value compared to a comparable reference. In some embodiments, the term "reduced" or its equivalent means complete or substantially complete suppression, i.e., a reduction to zero or substantially to zero. In some embodiments, the term "increased" or "induced" means an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher in the level of the assessed value compared to a comparable reference.

[0347] "In sequence": As used in this article for polynucleotides or polynucleotides, "in sequence" refers to the order of features along the polynucleotide or polynucleotide from 5' to 3'. As used in this article for polypeptides, "in sequence" refers to the order of features along the polypeptide from the feature closest to the N-terminus to the feature closest to the C-terminus. "In sequence" does not mean that other features cannot be present among the listed features. For example, if features A, B, and C of a polynucleotide are described in this article as "feature A, feature B, and feature C in sequence," this description does not exclude, for example, feature D being located between features A and B.

[0348] Ionizable: The term "ionizable" refers to a compound, group, or atom that is charged at a given pH. In the context of ionizable amino lipids, such lipids or their functional groups or atoms carry a positive charge at a given pH. In some embodiments, ionizable amino lipids are positively charged at acidic pH. In some embodiments, ionizable amino lipids are primarily neutral at physiological pH values ​​(e.g., about 7.0-7.4 in some embodiments), but become positively charged at lower pH values. In some embodiments, ionizable amino lipids may have a pKa in the range of about 5 to about 7.

[0349] Isolated: The term "isolated" means altered from or removed from its native state. For example, nucleic acids or peptides naturally present in living animals are not "isolated," but the same nucleic acids or peptides that are partially or completely separated from their native coexisting material are "isolated." Isolated nucleic acids or proteins may exist in a substantially purified form or may exist in non-native environments (e.g., host cells).

[0350] Lipids: As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules comprising one or more hydrophobic portions or groups and optionally one or more hydrophilic portions or groups. Molecules comprising both hydrophobic and hydrophilic portions are also commonly referred to as amphiphilic molecules.

[0351] RNA lipid nanoparticles: As used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and an RNA molecule (e.g., one or more polynucleotides as provided herein). In some embodiments, the RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, the RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one accessory lipid, and at least one polymer-conjugated lipid (e.g., a PEG-conjugated lipid). In various embodiments, the RNA lipid nanoparticles as described herein may have an average size (e.g., Z-mean) of about 100 nm to 1000 nm, about 200 nm to 900 nm, about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of this disclosure, the RNA lipid nanoparticles may have a particle size (e.g., Z-mean) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, the average size of the lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, the RNA lipid nanoparticles may be prepared by mixing lipids with the RNA molecules described herein.

[0352] Neutralization: As used herein, the term "neutralization" refers to an event in which a binder (such as an antibody) binds to the biologically active site of a virus (such as a receptor-binding protein), thereby inhibiting parasitic infection of the cell. In some embodiments, the term "neutralization" refers to an event in which the binder eliminates or significantly reduces the ability to infect cells.

[0353] Nucleic Acids / Polynucleotides: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, the nucleic acid is or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, the nucleic acid is or comprises a single-stranded nucleic acid. In some embodiments, the nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, the nucleic acid comprises both a single-stranded portion and a double-stranded portion. In some embodiments, the nucleic acid comprises a backbone containing one or more phosphodiester bonds. In some embodiments, the nucleic acid comprises a backbone containing both phosphodiester bonds and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may comprise a backbone containing one or more phosphate thioester or 5'-N-phosphamide bonds and / or one or more peptide bonds, as in "peptide nucleic acid". In some embodiments, the nucleic acid comprises one or more or all of the natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all of the non-natural residues. In some embodiments, the non-natural residues comprise nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5-propynyl-cytidine, C-5-propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazoadenosine, 7-deazoguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, compared to the sugar in the natural residues, the non-natural residues comprise one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product (such as RNA or a polypeptide). In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolating from a natural source, enzymatically synthesizing (e.g., by polymerase based on a complementary template, e.g., in vivo or in vitro), replicating in a recombinant cell or system, or chemically synthesizing.In some implementation schemes, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 800 0, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0354] Pharmaceutically Effective Amount: The term "pharmaceutically effective amount" or "therapeuticly effective amount" refers to the amount, alone or in combination with a larger dose, that achieves the desired response or effect. In the case of treating a specific disease (e.g., HIV), the desired response in some embodiments involves the process of suppressing the disease (e.g., HIV). In some embodiments, such suppression may include slowing the progression of the disease (e.g., HIV) and / or interrupting or reversing the progression of the disease (e.g., HIV). In some embodiments, the desired response in the treatment of a disease (e.g., HIV) may be or may include delaying or preventing the onset of the disease (e.g., HIV) or disorder (e.g., HIV-related disorder). The effective amount of the composition (e.g., pharmaceutical composition) described herein will depend on factors such as, for example, the disease (e.g., HIV) or disorder to be treated (e.g., HIV-related disorder), the severity of such disease (e.g., HIV) or disorder (e.g., HIV-related disorder), the patient's individual parameters (including, for example, age, physical condition, body size, and weight), the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Therefore, the dosage of the compositions described herein (e.g., pharmaceutical compositions) can depend on various such parameters. In cases where the patient's response to the initial dose is insufficient, a higher dose may be used (or an effective higher dose may be obtained through a different, more localized route of administration).

[0355] Polypeptide: As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that is naturally occurring. In some embodiments, the polypeptide has an amino acid sequence that is not naturally occurring. In some embodiments, the polypeptide has an engineered amino acid sequence because it is designed and / or generated through artificial means. In some embodiments, the polypeptide may comprise natural amino acids, non-natural amino acids, or both, or be composed of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide may comprise only natural amino acids, or only non-natural amino acids, or be composed of only those. In some embodiments, the polypeptide may comprise D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may comprise only D-amino acids. In some embodiments, the polypeptide may comprise only L-amino acids. In some embodiments, the polypeptide may include one or more side groups or other modifications, such as modifications or attachments to one or more amino acid side chains at the N-terminus, the C-terminus, or any combination thereof. In some embodiments, such side groups or modifications include acetylation, amidation, esterification, methylation, polyethylene glycolation, etc., including combinations thereof. In some embodiments, the polypeptide may be cyclic and / or may contain a cyclic moiety. In some embodiments, the polypeptide is not cyclic and / or does not contain any cyclic moiety. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or contain a pinned polypeptide. In some embodiments, the term "polypeptide" may be attached to the name, activity, or structure of a reference polypeptide; in such cases, it is used herein to refer to polypeptides that share a common associated activity or structure and can therefore be considered members of the same class or family of polypeptides. For each such class, this specification provides and / or those skilled in the art will recognize exemplary polypeptides within that class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides of that polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of that class; in some embodiments, they share common sequence motifs (e.g., characteristic sequence elements) and / or common activities (in some embodiments, at comparable levels or within specified ranges).For example, in some embodiments, the member polypeptide exhibits at least about 30-40% homology or identity with the overall sequence of the reference polypeptide, and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and / or includes at least one region (e.g., in some embodiments, this may be or contain a conserved region of characteristic sequence elements) that exhibits very high sequence identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%. Such conserved regions typically encompass at least 3-4 and usually up to 35 or more amino acids; in some embodiments, the conserved region encompasses at least one segment containing at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more adjacent amino acids. In some embodiments, the associated polypeptide may comprise or consist of a fragment of the parent polypeptide.

[0356] Prevention: As used herein, the term "prevention" when used in connection with the occurrence of a disease, condition, and / or disorder refers to reducing the risk of developing such disease, condition, and / or disorder and / or delaying the onset of one or more features or symptoms of such disease, condition, or disorder. Prevention can be considered complete when the onset of a disease, condition, or disorder has been delayed for a predefined period of time.

[0357] Reference: As used herein, the term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. Generally, as those skilled in the art will understand, a reference or control is determined or characterized under conditions or circumstances comparable to those conditions or circumstances being evaluated. Those skilled in the art will understand when sufficient similarity exists to justify reliance on a particular possible reference or control and / or comparison with a particular possible reference or control.

[0358] Ribonucleic acid (RNA) or polynucleotide: As used herein, the terms “ribonucleic acid,” “RNA,” or “polynucleotide” refer to a polymer of ribonucleotides. In some embodiments, the RNA is single-stranded. In some embodiments, the RNA is double-stranded. In some embodiments, the RNA comprises both single-stranded and double-stranded portions. In some embodiments, the RNA may comprise a backbone structure as described in the definition of “nucleic acid / polynucleotide” above. The RNA may be regulatory RNA (e.g., siRNA, microRNA, etc.) or messenger RNA (mRNA). In some embodiments, the RNA is mRNA. In some embodiments where the RNA is mRNA, the RNA typically includes a poly(A) region at its 3' end. In some embodiments where the RNA is mRNA, the RNA typically includes a cap structure recognized in the art at its 5' end, for example, for recognizing the mRNA and ligating it to a ribosome to initiate translation. In some embodiments, the RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic synthesis and / or by chemical synthesis).

[0359] Ribonucleotides: As used herein, the term "ribonucleotide" encompasses both unmodified and modified ribonucleotides. For example, unmodified ribonucleotides include purine bases adenine (A) and guanine (G) and pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications, including but not limited to, for example, (a) end modifications, such as 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse bonding, etc.), 3' end modifications (e.g., conjugation, reverse bonding, etc.), (b) base modifications, such as substitution with a modified base, a stabilizing base, a destabilizing base, or a base paired with or conjugated with an extended partner library base, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleotide linking modifications, including modifications or substitutions of phosphodiester links. The term "ribonucleotide" also covers ribonucleotide triphosphates, including both modified and unmodified ribonucleotide triphosphates.

[0360] Risk: As will be understood from the context, “risk” for a disease, condition, and / or disorder refers to the likelihood that a particular individual will develop the said disease, condition, and / or disorder. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is expressed as risk relative to the risk associated with a reference sample or reference sample group. In some embodiments, the reference sample or reference sample group has a known risk of a disease, condition, disorder, and / or event. In some embodiments, the reference sample or reference sample group is from individuals comparable to the particular individual. In some embodiments, risk may reflect one or more genetic attributes, such as those that may predispose an individual to (or prevent) a particular disease, condition, and / or disorder. In some embodiments, risk may reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes.

[0361] Selectivity or Specificity: The terms “selectivity” or “specificity” as used herein to refer to an active agent are understood by those skilled in the art to mean that the agent distinguishes potential target entities, states, or cells. For example, in some embodiments, an agent is considered to bind “specifically” to its target if it preferentially binds to its target in the presence of one or more competing alternative targets. In many embodiments, specific interactions depend on the presence of specific structural features of the target entity (e.g., epitopes, clefts, binding sites). It is to be understood that specificity is not necessarily absolute. In some embodiments, specificity can be assessed relative to the specificity of the target-binding portion to one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding portion. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding portion.

[0362] Substituted or Optionally Substituted: As described herein, the compounds of the present invention may contain an "optionally substituted" portion. Generally, regardless of whether the preceding term "optionally" is used, the term "substituted" means that one or more hydrogens of the specified portion are replaced by suitable substituents. "Substituted" applies to one or more hydrogens explicitly or implicitly present in the structure (e.g., At least refers to and At least refers to Unless otherwise indicated, an "optionally substituted" group may have suitable substituents at each substituted position of the group, and the substituents at each position may be the same or different when more than one position in any given structure may be substituted by more than one substituent selected from the specified group. The combinations of substituents contemplated by the present invention are preferably those that result in the formation of stable or chemically viable compounds. As used herein, the term "stable" means a compound that remains substantially unchanged when subjected to conditions of permissible manufacture, testing, and in some embodiments, recovery, purification, and use for one or more purposes provided herein. A group described as "substituted" preferably has between one and four substituents, more preferably one or two substituents. A group described as "optionally substituted" may be unsubstituted or "substituted" as described above.

[0363] The suitable monovalent substituent on the substituted carbon atom of the "optionally substituted" group is independently a halogen; -(CH2) 0- 4R o ;-(CH2) 0-4 OR o ;-O(CH2) 0-4 R o -O-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 CH(OR o )2;-(CH2) 0-4 SR o ;-(CH2) 0-4 Ph, which can be R o Substitution; -(CH2) 0-4 O(CH2) 0-1 Ph, which can be R o Substitution; -CH=CHPh, which can be replaced by R o Substitution; -(CH2) 0-4 O(CH2) 0-1 -pyridyl group, which can be R o Substitution; -NO2; -CN; -N3; ​​-(CH2) 0-4 N(R o )2;-(CH2) 0- 4N(R o )C(O)R o ;-N(R o )C(S)R o ;-(CH2) 0-4 N(R o )C(O)NR o 2; -N(R) o )C(S)NR o2;-(CH2) 0-4 N(R o )C(O)OR o ;-N(R o )N(R o )C(O)R o ;-N(R o )N(R o )C(O)NR o 2;-N(R o )N(R o )C(O)OR o ;-(CH2) 0-4 C(O)R o ;C(S)R o ;-(CH2) 0-4 C(O)OR o ;-(CH2) 0-4 C(O)SR o ;-(CH2) 0-4 C(O)OSiR o 3;-(CH2) 0-4 OC(O)R o ;-OC(O)(CH2) 0-4 SR o ;-(CH2) 0-4 SC(O)R o ;-(CH2) 0-4 C(O)NR o 2;-C(S)NR o 2;-C(S)SR o ;-SC(S)SR o 、-(CH2) 0- 4OC(O)NR o 2;-C(O)N(OR o )R o ;-C(O)C(O)R o ;-C(O)CH2C(O)R o ;-C(NOR o )R o ;-(CH2) 0-4 SSR o ;-(CH2) 0-4 S(O)2R o ;-(CH2) 0-4 S(O)2OR o ;-(CH2) 0-4 OS(O)2R o ;-S(O)2NR o 2;-(CH2) 0-4 S(O)Ro ;-N(R o )S(O)2NR o 2; -N(R) o )S(O)2R o ;-N(OR) o )R o ;-C(NH)NR o 2; -P(O)2R o ;-P(O)R o 2; -OP(O)R o 2; -OP(O)(OR o )2; SiR o 3; -(C 1-4 (linear or branched alkylene)ON(R) o )2; or -(C 1-4 (straight-chain or branched-chain alkylene)C(O)ON(R) o )2, where each R o It can be replaced and independently of halogen, C as defined below. 1-6 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph, -CH2- (5- to 6-membered heteroaryl ring) or 3- to 6-membered saturated, partially unsaturated or aryl ring having 0 to 4 independent heteroatoms selected from nitrogen, oxygen or sulfur, or, although defined above, two independently occurring R... o Together with its intercalary atoms, it forms a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring having 0-4 independent heteroatoms selected from nitrogen, oxygen, or sulfur, wherein the monocyclic or bicyclic ring may be substituted as defined below.

[0364] In R o Appropriate monovalent substituents (or R that makes two independent occurrences) o The ring formed by the intercalary atoms is independently a halogen, –(CH2). 0–2 R ● –(halogenated R) ● ), –(CH2) 0–2 OH, –(CH2) 0–2 OR ● –(CH2) 0–2 CH(OR ● 2. -O(halogenated R) ● –CN, –N3, –(CH2) 0–2 C(O)R ● –(CH2) 0–2 C(O)OH, –(CH2) 0–2 C(O)OR ● –(CH2) 0–2 SR● –(CH2) 0–2 SH, –(CH2) 0–2 NH2、–(CH2) 0–2 NHR ● –(CH2) 0–2 NR ● 2. –NO2, –SiR ● 3. –OSiR ● 3. -C(O)SR ● 、–(C 1–4 (straight-chain or branched-chain alkylene)C(O)OR ● Or –SSR ● , where each R ● It is either unsubstituted or, in the case of a preceding "halogen group," substituted with only one or more halogens, and independently selected from C. 1–4 Aliphatic group, –CH2Ph, –O(CH2) 0–1 Ph or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. R o Suitable divalent substituents on saturated carbon atoms include =O and =S.

[0365] Suitable divalent substituents on the saturated carbon atom of the "optionally substituted" group include the following: =O ("oxo"), =S, =NNR. * 2、=NNHC(O)R * =NNHC(O)OR * =NNHS(O)2R * =NR * =NOR * 、 –O(C(R) * 2)) 2– 3O – or –S(C(R) * 2)) 2–3 S–, where each independently occurring R * Selected from hydrogen, and C that can be substituted as defined below. 1–6 Aliphatic group or unsubstituted 5- to 6-membered saturated, partially unsaturated, or aromatic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents bonded to the adjacent substituted carbon of the "optionally substituted" group include: –O(CR * 2) 2–3 O–, where each independently occurring R * Selected from hydrogen, and C that can be substituted as defined below. 1–6 Aliphatic group or unsubstituted 5–6 saturated, partially unsaturated or aromatic ring having 0–4 heteroatoms independently selected from nitrogen, oxygen or sulfur.

[0366] R* Suitable substituents on aliphatic groups include halogens, -R ● -(halogenated R) ● -OH, -OR ● –O(halogenated R) ● ), –CN, –C(O)OH, –C(O)OR ● –NH2, –NHR ● –NR ● 2 or –NO2, where each R ● It is either unsubstituted or, in the case of a preceding "halogen group," substituted by only one or more halogens, and is independently C. 1–4 Aliphatic groups, –CH2Ph, –O(CH2) 0– 1Ph or a 3- to 6-membered saturated, partially unsaturated, or aromatic ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0367] Suitable substituents on the substituted nitrogen of the "optionally substituted" group include Each of them Independently, it is hydrogen, or C that is substituted as defined below. 1–6 Aliphatic group, unsubstituted –OPh, or a 3- to 6-membered saturated, partially unsaturated, or aromatic ring having 0-4 independently selected heteroatoms chosen from nitrogen, oxygen, or sulfur, or, although defined above, two independently occurring... Together with its intercalary atoms, it forms a 3- to 12-membered saturated, partially unsaturated, or aryl monocyclic or bicyclic ring with 0 to 4 independent heteroatoms selected from nitrogen, oxygen, or sulfur.

[0368] Suitable substituents on the aliphatic group are independently halogens, -R ● -(halogenated R) ● -OH, -OR ● -O(halogenated R) ● -CN, -C(O)OH, -C(O)OR ● -NH2, -NHR ● -NR ● 2 or -NO2, where each R ● It is either unsubstituted or, in the case of a preceding "halogen group," substituted by only one or more halogens, and is independently C. 1-4 Aliphatic groups, -CH2Ph, -O(CH2) 0-1 Ph or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0 to 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0369] Subject: As used herein, the term "subject" refers to an organism to which the composition described herein is to be administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject has a disease, condition, or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, the subject is susceptible to a disease, condition, or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, condition, or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, the subject exhibits one or more nonspecific symptoms of a disease, condition, or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, condition, or disorder (e.g., HIV, HIV-related disorders, etc.). In some implementations, the subject is someone who has a predisposition to a disease, condition, or disorder (e.g., HIV, HIV-related disorders, etc.) or one or more characteristics characteristic of the aforementioned risks. In some implementations, the subject is a patient. In some implementations, the subject is an individual who has received and / or has received diagnostic and / or therapeutic treatments.

[0370] "Having": An individual who has been diagnosed with and / or exhibits one or more symptoms of a disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.)

[0371] Susceptible: An individual with a "susceptible" disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.) is an individual at a higher risk of developing the aforementioned disease, condition, and / or disorder than members of the general public. In some embodiments, an individual with a susceptible disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.) may not yet be diagnosed with the aforementioned disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, an individual with a susceptible disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.) may exhibit symptoms of the aforementioned disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, an individual with a susceptible disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.) may not exhibit symptoms of the aforementioned disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.). In some implementations, individuals susceptible to a disease, condition, and / or illness (e.g., HIV, HIV-related illnesses, etc.) will develop the disease, condition, and / or illness (e.g., HIV, HIV-related illnesses, etc.). In some implementations, individuals susceptible to a disease, condition, and / or illness (e.g., HIV, HIV-related illnesses, etc.) will not develop the disease, condition, and / or illness (e.g., HIV, HIV-related illnesses, etc.).

[0372] Therapy: The term "therapy" refers to the administration or delivery of an agent or intervention that has a therapeutic effect and / or induces the desired biological and / or pharmacological effect (e.g., has been shown to be statistically likely to have such an effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to reduce, improve, alleviate, suppress, prevent, or treat one or more symptoms or characteristics of a disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.), delay its onset, reduce its severity, and / or reduce its incidence. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to reduce, alleviate, suppress, prevent, or treat one or more symptoms or characteristics of a disease, condition, and / or disorder, delay its onset, reduce its severity, and / or reduce its incidence.

[0373] Treatment: As used herein, the term "treatment" refers to any method used to partially or completely reduce, improve, alleviate, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.). Treatment may be administered to subjects who do not exhibit symptoms of a disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.). In some embodiments, treatment may be administered to subjects exhibiting only early symptoms of a disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.), for example, for the purpose of reducing the risk of developing a pathology associated with said disease, condition, and / or disorder. In some embodiments, treatment may be administered to subjects in a late stage of a disease, condition, and / or disorder (e.g., HIV, HIV-related disorders, etc.). Detailed Implementation

[0374] I. Human Immunodeficiency Virus (HIV)

[0375] Human immunodeficiency virus (HIV) is a lentivirus belonging to the retroviridae family. Mature HIV particles are typically spherical and approximately 100 nm in diameter. They consist of a core (from innermost to outermost) containing two identical single-stranded RNA molecules, a capsid, and an envelope. Figure 1 (Figure B) (Musumeci et al., Molecules 20.9(2015):17511-17532, which is incorporated herein by reference). The envelope consists of a lipid bilayer and Env proteins. These Env proteins exist as trimers of the gp120 surface protein and are anchored to the envelope by the gp41 transmembrane protein. The viral capsid is surrounded by the envelope and comprises a symmetrical outer capsid membrane composed of the matrix protein p17. Inside the outer capsid membrane is a cone-shaped capsid containing the inner capsid protein p24. The inner capsid attaches to the outer capsid membrane at its cone-shaped portion. The inner capsid contains viral RNA (two identical copies) and viral enzymes: reverse transcriptase, integrase, and protease. The virus particles also contain oligopeptides produced during the maturation of the virus particles by the proteolytic processing of Gag and Gag / Pol precursor proteins p55 and p160 (GAC, Transfusion Medicine Hemotherap y,43:203–222,2016, which is incorporated herein by reference).

[0376] There are two main types of HIV: HIV-1 and HIV-2. HIV-1 is the most common type of HIV, accounting for 95% of all infections worldwide. HIV-2 is relatively rare and less infectious. HIV-2 is mainly concentrated in West Africa and surrounding countries.

[0377] HIV-1 and HIV-2 share many similarities, including their intracellular replication pathways, modes of transmission, and clinical outcomes leading to acquired immunodeficiency syndrome (AIDS). However, due to the lower transmissibility of HIV-2, it is less likely to progress to AIDS. Therefore, individuals infected with HIV-2 typically experience no visible disease progression over extended periods, while those infected with HIV-1 tend to progress more rapidly and are more susceptible to AIDS.

[0378] However, once progression begins, the pathological processes of the two viruses are largely similar. One difference is that HIV-2 has been found to progress at higher CD4 counts. Furthermore, HIV-2 infection is characterized by a lower viral load of over 10,000 copies / mL compared to the millions of copies / mL of HIV-1. In cases of HIV-2 infection, the immune response in subjects tends to be more protective, thus slowing disease progression.

[0379] HIV-1 and HIV-2 are further divided into groups and subtypes. HIV-1 is divided into the major or M group, the aberrant or O group, and the non-M / O or N group. The most common group is the M group, which is primarily responsible for the global HIV epidemic. Other groups are relatively rare and are found in selected geographical locations, including Gabon, Cameroon, and Equatorial Guinea.

[0380] Group M is further divided genetically into different subtypes: A, B, C, D, F, G, H, J, and K. Some of these subtypes combine to form a hybrid virus known as "circular recombination." Globally, subtype B accounts for 12% of HIV infections. Subtype B is the dominant HIV-1 subtype found in the Americas, Australia, and Western Europe. Therefore, most HIV clinical research to date has focused on this population.

[0381] Although subtype C accounts for nearly 50% of all HIV infections, research on this subtype is limited. Subtype C is common in southern African countries, where HIV prevalence is high. Cameroon and the Democratic Republic of Congo are the origin regions of HIV-1, exhibiting rich HIV-1 subtype diversity. However, due to population mixing and migration, global subtype distribution patterns are now changing.

[0382] Approximately eight HIV-2 subtypes have been identified to date. The two main prevalent HIV-2 subtypes are A and B. HIV-2 group A infection is predominantly found in West Africa, but a few cases have also been reported in Brazil, Europe, the United States, and India. HIV-2 group B infection is found only in West Africa.

[0383] Because HIV subtypes can be geographically diverse, an ideal therapeutic agent would target and neutralize more than one subtype, or even more preferably multiple HIV strains. As discussed further below, anti-HIV antibodies capable of binding to and at least temporarily neutralizing HIV viral particles have been developed. Nevertheless, such anti-HIV antibodies still present challenges, including administration difficulties, antibody persistence in the body, and viral escape. As described herein, the polynucleotides and compositions disclosed address these challenges.

[0384] A. HIV genome

[0385] HIV contains two identical copies of a single-stranded DNA that encodes its genome. When the virus integrates into a host cell, the viral RNA is reverse transcribed into double-stranded DNA, resulting in RNA degradation and integration of the double-stranded DNA or proviral DNA into the host genome. The HIV genome is flanked at both ends by LTR (long terminal repeat) regions, including the 5' LTR that encodes the transcription promoter. The RNA genome is 9749 nucleotides and contains a 5' cap, a 3' poly(A) tail, and several open reading frames (ORFs) (Wain-Hobson et al., Cell 40(1):9-17, 1985, which is incorporated herein by reference).

[0386] The HIV genome includes the following genes: gag, pol, vif, vpr, tat, rev, vpu, env, and nef (see also: Figure 1 (Figure A). The proteins encoded by gag, pol, and env are viral structural proteins. The proteins encoded by tat and rev are essential regulatory proteins. The proteins encoded by nef, vpr, vif, and vpu are accessory regulatory proteins. The gag gene encodes the P555Gag precursor protein of the outer core membrane protein (p17), capsid protein (p24), nucleoprotein (p7), Pr55Gag, and p6. Protein p24 forms the cone-shaped capsid, and protein p17 forms the inner membrane layer. Protein p6 is involved in viral particle release.

[0387] The pol gene encodes the Pr160GagPol precursor protein, protease p10, reverse transcriptase (p51), and RNase H (p15), or both together as the p66 protein, and integrase p32. Pr160GagPol is the precursor of viral enzymes p10, p51, and p15. The proteolytic cleavage of Gag (Pr55) and Gag-Pol (Pr160GagPol) produces protease p10. The p51 reverse transcriptase is responsible for transcribing HIV RNA into proviral DNA. When proviral DNA is produced, the p55 protein (RNase H) is used to degrade viral RNA in the viral RNA / DNA complex. The p32 integrase protein integrates the proviral DNA into the host cell genome.

[0388] The env gene encodes PrGp160, a precursor protein to two envelope glycoproteins: gp120 (surface protein) and gp41 (transmembrane protein). Proteins gp120 and gp41 are produced by the protease cleavage of the precursor protein PrGp160. The role of protein gp120 is to attach the virus to the target host cell. Protein gp41 anchors gp120 to the viral membrane and is used for fusion of the virus and the target cell membrane.

[0389] The gene `tat` encodes the Tat protein p14 (a transactivator protein), which activates the transcription of viral genes. The gene `rev` encodes the Rev protein p19 (an RNA splicing regulator), which regulates the output of mRNA (both non-splicing and partially splicing). The gene `nef` encodes the Nef protein p27 (a negative regulator), which plays a role in HIV replication and enhances viral infectivity in host cells. Protein p27 also downregulates CD4 and HLA on target cells. The gene `vif` encodes the Vif protein p23 (a viral infection factor), which functions to produce the virus in host cells. The gene `vpr` encodes the Vpr protein p15 (a viral protein r). This protein interacts with the p6 protein and promotes viral infectivity in host cells. The gene `vpu` encodes the Vpu protein p16 (a virus-specific protein), which allows for the efficient release of viral particles and controls CD4 degradation on target cells. Protein p16 also controls intracellular signaling. The gene vpx encodes the Vpx protein p15 (viral protein x), which interacts with the p6 protein and is important in the early stages of viral replication. The gene tev encodes the Tat / Rev protein p26, which is a fusion protein that regulates the Tat and Rev proteins (GAC, Transfusion Medicine Hemotherapy, 43:203-222, 2016, which is incorporated herein by reference).

[0390] B. Life Cycle

[0391] The HIV life cycle involves HIV viral particle entry into target host cells, reverse transcription of the viral genome, integration into the host genome, and protein maturation. To initiate infection, HIV particles contact the target host cell. The surface glycoprotein env gp120 of the mature HIV particle binds to the CD4 receptor on the target host cell, initiating additional binding of gp120 to co-receptors (i.e., chemokine receptor 5 (CCR5) or chemokine receptor 4 (CXCR4 of the fusionin)). Binding of gp120 to CD4 and the co-receptors triggers a conformational change in gp120, allowing gp41 to be presented on the viral membrane and fuse with the plasma membrane of the target host cell. The viral capsid then enters the cytoplasm of the host cell. The capsid is absorbed by endosomes, releasing its contents, namely viral RNA. After entering and being released into the target host cell, the virus undergoes reverse transcription, in which the viral RNA is reverse transcribed into single-stranded cDNA. Next, the RNA strand is degraded by RNase H, and the single-stranded cDNA is converted into double-stranded DNA by the DNA-dependent DNA polymerase activity of reverse transcriptase.

[0392] Double-stranded DNA or proviral DNA forms a complex with integrase and is transported to the host cell nucleus, where it randomly inserts into the host cell genome. Once integrated into the genome, the proviral genome replicates. The proviral genome can replicate along with the host cell genome as part of cell division, or it can replicate using its own mechanisms. For example, the LTR promoter creates an attachment site for cellular DNA-dependent RNA polymerases and transcription factors to initiate transcription. Tat proteins accelerate the transcription of proviral DNA.

[0393] The processes of entry into target host cells, reverse transcription, integration, and protein maturation can be completed in less than 24 hours, and progeny viral particles can be detected within 12 hours post-infection. The first progeny viral particles may be released from infected cells approximately 24 hours after infection. Infected T cells are typically eliminated by the immune system (e.g., by cytotoxic T cells) at a rate of 2–4 days. Due to the destruction of HIV-infected T cells and the restriction of T cell production, helper T cells are reduced. The proteins nef and tat also inhibit the maturation and replacement of helper T cells. Therefore, over time, HIV infection will lead to immunodeficiency (GAC, Transfusion Medicine Hemotherapy, 43:203-222, 2016, which is incorporated herein by reference).

[0394] C. Transmission and Pathology

[0395] HIV enters the body through intact mucous membranes, damaged skin, or through parenteral inoculation. HIV is most commonly transmitted sexually. HIV can be detected systemically approximately 10–14 days after infection, and transmission through blood or transplanted organs can occur approximately 5–6 days later. Clinical symptoms typically appear 3–6 weeks after infection and may include fever, swollen lymph nodes, fatigue, rash, gastrointestinal symptoms, acute neuropathy, myalgia, and / or malaise. However, some individuals are asymptomatic during this acute phase. These symptoms of acute or primary infection can last 2–6 weeks. Following this initial symptomatic period is usually an asymptomatic or period of occasional symptoms, which can last for years.

[0396] Without treatment, HIV infection leads to progressive loss of CD4+ T cells, which can cause a range of immune abnormalities and increase the risk of infections and cancer complications. In addition, HIV infection can cause cardiovascular disease, bone disease, kidney and liver dysfunction, and several other common conditions.

[0397] Although antiviral therapy (ART) has been developed to treat HIV infection, ART can only prevent new cells from becoming infected; that is, if a cell already contains viral DNA integrated into its genome, ART cannot eliminate the infection. Furthermore, HIV establishes a latent infection in CD4+ T cells, which can persist indefinitely, and some have the ability to self-renew. Once HIV integrates into the cell's genome, it can potentially restart replication within the cell. (Deeks et al., Nature Reviews 1.1 2015 and GAC, Transfusion Medicine Hemotherapy, 43:203-222, 2016, which are incorporated herein by reference).

[0398] D. Treatment Strategies

[0399] The development of targeted HIV therapies faces many challenges. One challenging factor is viral heterogeneity. HIV can be divided into at least two main types (HIV-1, found globally; and HIV-2, primarily in West Africa), with HIV-1 further subdivided into three subgroups (M, N, O, and P), and M further subdivided into the subtype AL. Subtypes can recombine after co-infection, resulting in other recombinant subtypes.

[0400] Another challenging factor is the high mutation rate of HIV in vivo. A recent study quantified the spontaneous mutation rate of HIV-1 across the entire genome in the DNA sequence of peripheral blood mononuclear cells and revealed an extremely high mutation rate per base per cell (4.1 ± 1.7) × 10⁻⁶. -3This is the highest mutation rate reported for any biological entity (Cuevas et al., PloS Biol 2015, which is incorporated herein by reference). Therefore, the ability to identify and develop therapeutics targeting conserved epitopes in multiple groups and subtypes of the constantly mutating HIV sequence is extremely challenging, especially given the virus's unique ability to evade the immune system.

[0401] In addition to its high mutation frequency, HIV poses other challenges to the immune system, making its treatment uniquely challenging. Therapeutic targets on HIV include the HIV envelope protein (HIV Env); however, HIV Env is heavily glycosylated, and the Env site is therefore shielded from treatment by the presence of glycans. Furthermore, Env glycans are host-derived and can be highly heterogeneous.

[0402] Recent treatment strategies involve the use of broad-spectrum neutralizing antibodies (bNAbs), which are antibodies capable of neutralizing a wide range of HIV isolates worldwide. These antibodies have been identified in HIV-infected individuals considered “elite neutralizers,” representing <10% of all HIV patients (Burton and Hangartner, Ann. Rev. Immunol. 2016, which is incorporated herein by reference). These antibodies provide insights into potential target epitopes and structures for therapeutic agents. Other advances contributing to therapeutic progress include the generation of stable HIV Env spike trimers (Sanders and Moore, Immunol. Rev. 2017, which is incorporated herein by reference) and the characterization of their structures at high resolution (Ward and Wilson, Immunol. Rev. 2017, which is incorporated herein by reference). Examples of potential Env sites include apical sites, high-mannose plaques in the gp120 region, the gp120-gp41 interface region, the gp41 proximal membrane region (MPER), and CD4 binding sites (see [link to Env site]). Figure 2 (From McCoy and Burton, Immunol Rev. 275.111-20 2017, which is incorporated herein by reference). These sites each present unique challenges as therapeutic targets for bnAbs. For example, bnAbs targeting the gp41-gp120 interface must be able to bind to complex heteroglycans. BnAbs targeting the Env protein CD4 binding site have been found to exhibit high levels of somatic hypermutation.

[0403] Nevertheless, among these sites, the apical site is of particular interest because it is a conserved region identified in the HIV envelope trimer (Env).

[0404] As discussed in this paper, the HIV-1 envelope (Env) glycoprotein trimer is a trimer of the gp120-gp41 heterodimer, the only viral protein on the surface of the HIV-1 virus, and therefore an important target of bnAb. The apex of the Env trimer includes variable 1 and 2 (V1V2) loops and a variable 3 (V3) loop. The V1V2 loop contains four β chains. Furthermore, the V1V2 epitope is of particular interest as a target because it is a region of gp120 that undergoes significant conformational changes upon binding to CD4. For example, the interaction between CD4 and gp120 stabilizes the open Env conformation, which can interact with chemokine receptors to induce additional conformational changes, ultimately leading to the insertion of the gp41 fusion protein into the target cell membrane. In the closed state, the three V1V2 regions of gp120 shield the V3 and helper receptor binding sites. Therefore, certain bNAb targeting the apex (e.g., targeting V1V2 epitopes) are characterized by their ability to prevent the opening of the Env trimer to expose the V3 loop and co-receptor binding site, thereby preventing the conformational changes that lead to viral-target host cell membrane fusion (see Wang, Haoqing et al., “Asymmetric recognition of HIV-1 Envelope trimer by V1V2 loop-targeting antibodies.” Elife 6(2017):e27389, which is incorporated herein by reference).

[0405] In addition, this disclosure provides polynucleotides encoding antibody agents (e.g., bNAB) that target a broader group of HIV variants and thus enable the treatment of more HIV patients. Furthermore, this disclosure provides compositions for delivering polynucleotides encoding antibody agents (e.g., bNAB) that target various HIV sequences.

[0406] 1. Antiretroviral therapy for HIV

[0407] Currently, HIV infection is primarily treated with antiretroviral therapy (ART). ART is a type of medication that reduces HIV replication, increases CD4 cell count, and lowers the risk of transmission in infected individuals. The World Health Organization (WHO) recommends initiating ART for all HIV-infected adults, regardless of clinical stage or CD4 cell count (Consolidated guidelines on HIV prevention, testing, treatment, service delivery and monitoring: recommendations for a public health approach. Geneva: World Health Organization; 2021, which is incorporated herein by reference). However, ART is not a curative treatment; if an infected individual stops taking ART, viremia (e.g., viral load) will rapidly rebound. The high mutation rate of HIV also constrains patients to strictly adhere to their therapy to avoid escape mutants and treatment failure. Therefore, ART is intended to be taken daily for life by infected subjects.

[0408] Several classes of FDA-approved ART treatments for HIV work through different mechanisms. Effective management of HIV infection typically involves a combination of at least three ARTs to address the complex pathogenicity of the disease. The most effective ART combination often varies from person to person (see, for example, Bhatti et al., Cureus 2016, which is incorporated herein by reference). Cihlar et al., Current opinion in virology, 2016 (incorporated hereby in full), reviewed the classes of ART drugs used to treat HIV.

[0409] Table 1: Exemplary categories of ART for the treatment of HIV, their mechanisms of action, and exemplary compounds in each category.

[0410]

[0411] 2. HIV antibody agents

[0412] In addition to ART, anti-HIV antibodies have also been developed. Using anti-HIV antibodies to treat HIV typically requires antibodies to possess specific characteristics, including safety, a favorable pharmacokinetic profile, high neutralizing activity, and broad neutralizing activity to effectively target the diversity present in HIV viral particles. As with other HIV treatments (including, for example, ART), viral escape from anti-HIV antibodies remains a significant challenge.

[0413] For example, Barouch et al. infected rhesus monkeys with SHIV-SF162P3 (Barouch et al., Nature 503:7475224-228, 2013), which is incorporated herein by reference in full. The rhesus monkeys were then treated with three monoclonal antibodies (mAbs): the N332 glycan-dependent mAb PGT121 and the CD4 binding site-specific mAbs 3BNC117 and b12. The mAbs were administered as a mixture on days 0 and 7 at 10 mg / kg each; as a mixture alone on day 0 at 10 mg / kg each; or as a combination of PGT121 and 3BNC117 alone at 10 mg / kg each. Transient viral suppression was observed until bNAb levels dropped below 10 μg / mL. The mAbs were also administered alone to the rhesus monkeys, and PGT121 alone caused rapid virological control, with most animals rebounding after 6–8 weeks. Rhesus monkeys receiving the combination of PGT121 and 3BNC117 received a second dose on day 105 after a rebound in viral levels. Viral resuppression was observed, although the control was not as durable as with the previous administration.

[0414] Shingai et al. described rhesus monkeys infected with SHIV. AD8EO (Shingai et al., Nature 503:7475 277-280, 2013, which is incorporated herein by reference in its entirety). Rhesus monkeys were then treated with 10-1074 and 3BNC117 mAb, alone or in combination. At 12 weeks post-vaccination, both antibodies, administered alone at 10 mg / kg, caused rapid viral suppression, but viral levels rebounded rapidly. Combined administration of the two antibodies to chronically infected animals resulted in a longer period of suppression and increased CD4+ T cell levels, although viral levels subsequently rebounded. In other studies, pretreatment of rhesus monkeys with either antibody alone was found to prevent viral infection. Single-genome analysis of the rebounding virus in 10-074-treated rhesus monkeys revealed a mutation eliminating the gp120 N332 glycan, resulting in resistance to the mAb. However, SGA analysis of rebound virus in rhesus monkeys treated with 10-074 and 3BNC117 revealed that not all viruses contained in the rhesus monkeys underwent changes that conferred mAb resistance.

[0415] Caskey et al. described a first-in-human dose-escalation phase 1 clinical trial of 3BNC117 (CD4 binding site antibody) (Caskey et al., Nature 522.7557:487-491, 2015, which is incorporated herein by reference in full). Both uninfected and HIV-1-infected individuals participated in the trial. 3BNC117 at doses of 1, 3, 10, or 30 mg / kg was found to be generally safe and well-tolerated; no grade 3, 4, or serious adverse events were observed. Antibody clearance was observed to be faster in HIV-1-infected individuals than in uninfected controls. The effect of treatment on viral load was dose-dependent; doses of 10 and 30 mg / kg reduced viral load by up to 2.5 log. Viral resistance was observed in some individuals regardless of mAb dose, but not in others. The virus was cloned and sequenced; G459D was the mutation commonly observed in the 10 mg / kg group, and other mutations showed longer V5 loops (other mutations are described). Both mutations can alter sensitivity to CD4b.

[0416] Caskey et al. also evaluated 10-074, a potent mAb targeting the V3 loop of the HIV-1 envelope spike (Caskey et al., Nature Medicine 23.2:185-191, 2017), which is incorporated herein by reference in full. An open-label, phase 1, first-in-human clinical trial was conducted in 14 uninfected individuals and 19 HIV-1-infected individuals. Single intravenous infusions of 3, 10, or 30 mg / kg were administered. The mAb was found to be generally safe and well-tolerated; no grade 3, 4, or serious adverse events were observed. Antibody clearance was observed to be faster in HIV-1-infected individuals than in uninfected controls. Treatment suppressed viral load in individuals carrying susceptible 10-074 strains, followed by a rebound. Single-genome sequencing (SGS) of the rebound virus revealed that all patients who responded to treatment exhibited a PNGS at position N332 and an intact PNGS. 324 G(D / N)IR 327 Motif. Four weeks post-infusion, 91% of the envelope sequences contained amino acid mutations, with 97% of these mutations eliminating the PNGS at position 332 through mutations at N332 or S334. 3% of the mutated sequences... 324 G(D / N)IR 327A change was observed at D / N325 in the motif. Most mutations at the nucleic acid level were shifts, consistent with reverse transcriptase errors. Neutralization assays showed that the HIV-1 mutant resistant to 10-074 was not resistant to 3BNC117, VRC01, or PGDM1400 (mAbs targeting other regions of HIV-1). SGS performed one week post-infusion revealed that the resistant variants were either pre-existing or rapidly developed.

[0417] Bar et al. conducted two open-label laboratory trials investigating the safety, side effects, pharmacokinetic properties, and antiviral activity of VRC01 (a bNAb targeting the CD4 binding site of HIV) in patients who had interrupted antiretroviral therapy (ART) (Bar et al., New England Journal of Medicine 375.21:2037-2050, 2016, which is incorporated herein by reference in full). In one trial, the patient received three infusions of 40 mg / kg over a 6-week period; and in the other trial, eight infusions of 40 mg / kg over a 6-week period. Treatment was well tolerated, and no grade 3 or higher adverse events were observed. Neither trial produced durable suppression of plasma viremia, although a slightly longer rebound time was observed relative to historical controls. Regardless of rebound time, in one trial, resistance to VRC01 was almost universally increased in participants. Viral isolates in untreated samples showed stronger resistance to VRC01 neutralization compared to post-treatment. VRC01 treatment does not affect sensitivity to neutralization of other bNAbs.

[0418] Mendoza et al. conducted a phase 1b clinical trial evaluating the combination of 3BNC117 and 10-1074, administered at a dose of 30 mg / kg at weeks 0, 3, and 6 (Mendoza et al., Nature 561.7724:479-484, 2018, which is incorporated herein by reference in full). Both bNAbs target independent sites on the HIV-1 envelope spike. Infusions were generally found to be safe and well-tolerated, with no serious adverse events reported. The median rebound time following combination bNAb treatment was significantly prolonged. It was found that the two earliest rebounding individuals previously carried strains resistant to one or more bNAbs. Rebounded viruses clustered within a low-diversity lineage, consistent with the amplification (escape) of 1–2 relapsed viruses. Most rebounding viruses were found to contain the 10-1074 mutation, compared to the 3BNC117 mutation. However, combination bNAb therapy has demonstrated greater effectiveness than single bNAb treatment in curbing viral escape.

[0419] Gautam et al. on SHIV infection AD8EOThe results were analyzed in rhesus monkeys treated with 3BNC117-LS and 10-074-LS mAbs (Gautam, Rajeev et al., Nature Medicine 24.5:610-616, 2018, which is incorporated herein by reference in full). M428L and N343S (collectively referred to as LS) are mutations that increase the half-life in the mAb fragment domain. The LS mutation had no effect on virus neutralization in in vitro analyses. LS mAb was administered alone at 20 mg / kg and was well tolerated in all monkeys. The 10-1074-LS receptor showed increased protection against viral infection compared to the 3BNC117-LS receptor, but the LS mutation in both antibodies was more effective than WT. The decay rate of 10-1074-LS in serum was slower than that of 3BNC117-LS. mAb concentration / neutralizing activity was determined to predict the probability of infection. Experiments were performed with antibody pretreatment followed by viral challenge.

[0420] Schommers et al. characterized an anti-HIV antibody (referred to as “1-18”) in in vitro analysis and in HIV-1-infected humanized mice. 1-18 has been reported to bind to the CD4 binding site of HIV and to have potent potency and broad coverage against HIV strains. Schommers reported that 1-18 possesses some features previously found in other anti-HIV antibodies, which appear to contribute to the potency and broad coverage of 1-18: (1) 1-18 has an aromatic residue that mimics the CD4 residue Phe43 to target the “Phe43gp120 pocket”, a feature previously reported against anti-HIV antibody N6; (2) 1-18 contacts the adjacent gp120 primordium, as previously observed in anti-HIV antibody 3BNC117, but with an increased buried surface area (by inserting six of its residues into CDRH1); and (3) the buried surface area on gp120 is larger than that of other anti-HIV antibodies. Furthermore, it has been reported that 1-18 contacts conserved residues on HIV gp120 that are not accessed by other anti-HIV antibodies. Schommers hypothesized that these contacts might make 1-18 less dependent on classical CD4 binding site contact, thus making viral escape more difficult. Nevertheless, Schommers observed resistance to 1-18 in a minority of HIV strains.

[0421] Sok et al. reported that the PGDM1400 antibody showed superior potency against the 106 viral group compared to other bNAbs (e.g., PGT121, PGT128, and PGT151) (median IC50 was 0.003 μg / ml). Furthermore, the PGDM1400 antibody also showed high and broad (83% coverage) efficacy (see Sok, Devin et al., “Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex.” Proceedings of the National Academy of Sciences 111.49(2014):17624-17629; vander Velden, Yme U. et al., “Diverse HIV-1 escape pathways from broadly neutralizing antibody PGDM1400 in humanized mice.” Mabs. 12.1(2020)e1845908, which are incorporated herein by reference in their entirety).

[0422] In summary, the above data indicate that antibody administration can effectively treat or prevent HIV. However, based on the above studies and the following findings, it is evident that targeting such mutagenic viruses is difficult: (1) when a single broadly neutralizing antibody (bNAb) is used as a therapy, HIV develops resistance to the therapy within weeks (Bar et al., Effect of HIV Antibody VRC01 on Viral Rebound after Treatment Interruption, N. Engl. J. Med. 375, 2037–2050 (2016); Caskey et al., Viremia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117. Nature 522, 487–491 (2015); Caskey et al., Antibody 10–1074 suppresses viremia in HIV-1-infected individuals. Nat. Med. 23, 185–191 (2017); Klein et al., HIV therapy by a combination of broadly neutralizing antibodies in humanized mice, Nature). 492,118–122 (2012); Lynch et al., Virologic effects of broadly neutralizing antibody VRC01 administration during chronic HIV-1 infection, Sci. Transl. Med. 7, 319ra206 (2015); Scheid et al., HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption, Nature 535, 556–560 (2016), each of which is incorporated herein by reference in its entirety, and (2) certain antibody combinations improve viral control by preventing early development of resistance (Bar-On et al., Safety and antiviral activity of combination HIV-1 broadly neutralizing antibodies in viremic individuals, Nat. Med.).24, 1701–1707 (2018); Klein et al., 2012; Mendoza et al., Combination therapy with anti-HIV-1 antibodies maintains viral suppression, Nature 561, 479–484 (2018), each of which is incorporated herein by reference in its entirety. Viral rebound was observed in some of these antibodies, suggesting that they may only be effective for a limited time, such as before HIV escape mutations occur.

[0423] Therefore, there remains a need for therapeutic and preventative agents that can prevent viral escape and remain effective against HIV neutralization. As discussed herein, this disclosure provides techniques for administering polynucleotides encoding one or more antibody agents (e.g., anti-HIV antibody agents) to a subject. The techniques and methods described herein allow, for example, the simultaneous generation of different antibody agents from the polynucleotides. The antibody agents are designed to minimize or eliminate the risk of immunoglobulin chain mismatch. The ability to combine multiple antibody agent formulations as described herein (e.g., including the PGDM1400 antibody agent) allows for the development of a composition (e.g., a pharmaceutical composition) that delivers multiple antibody agents together such that they can bind to different epitopes of the HIV virus, thereby minimizing viral escape through mutations and increasing overall efficacy.

[0424] II. Polynucleotides for delivering antibody agents

[0425] In addition, this disclosure utilizes RNA technology as a novel method for directly expressing antibody agents in a subject, representing a novel antibody-based therapeutic approach. In some embodiments, as described herein, the polynucleotide encodes the immunoglobulin chain of the antibody agent.

[0426] In some embodiments, the antibody agent targets HIV. In some embodiments, the HIV-targeting antibody agent specifically binds to a specific epitope of the HIV peptide. For example, in some embodiments, the antibody agent specifically binds to an epitope comprising the tip or a portion of the Env trimer. See also Figure 2 From McCoy and Burton, Immunol Rev. 275.1 11-20, 2017, which is incorporated herein by reference.

[0427] In some implementations, the antibody agent may have at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10-9 A binding affinity (e.g., as measured by a dissociation constant) of M or lower for HIV epitopes (e.g., epitopes at the apex of the Env trimer). In some embodiments, the HIV antibody agent selectively binds to HIV target epitopes such that the binding between the HIV antibody agent and the target epitope is greater than 2, 5, 10, or 100 times greater than the binding between the HIV antibody agent and a non-target epitope. In some embodiments, the HIV antibody agent may have binding affinity for HIV epitopes and variants of said HIV epitopes. Those skilled in the art will understand that in some cases, binding affinity (e.g., as measured by a dissociation constant) may be affected by non-covalent intermolecular interactions, such as hydrogen bonding between two molecules, electrostatic interactions, hydrophobic forces, and van der Waals forces. Or, additionally, the binding affinity between a ligand and its target molecule may be affected by the presence of other molecules. Those skilled in the art will be familiar with various techniques for measuring affinity and / or dissociation constants according to this disclosure, including, but not limited to, ELISA, gel shift analysis, pull-down analysis, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), biolayer interferometry, grating-coupled interferometry, and spectroscopic analysis.

[0428] In some implementations, the HIV-targeting antibody may contain or be derived from a broad-spectrum neutralizing antibody (bNAb). In some implementations, the HIV-targeting antibody can be any of the following HIV-targeting antibodies: Sok et al., PNAS 111.49:17624-17629, 2014; van der Velden, Yme U. et al., Mabs.12.1:e1845908, 2020; Barouch et al., Nature 503:7475 224-228, 2013; Shingai et al., Nature 503:7475 277-280, 2013; Caskey et al., Nature 522.7557:487-491, 2015; Caskey et al., Nature Medicine 23.2:185-191, 2017; Bar et al., New England Journal of Medicine 375.21:2037-2050, 2016; Mendoza et al., Nature 561.7724:479-484, 2018; Gautam, Rajeev et al., Nature Medicine 24.5:610-616, 2018, the contents of which are incorporated herein by reference in their entirety for the purposes described herein.

[0429] In some embodiments, the HIV-targeting antibody may be, for example, 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, fragments thereof, or combinations thereof. Exemplary anti-HIV antibodies that can be used in the compositions described herein include, but are not limited to, 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, PGDM1400, fragments thereof, or combinations thereof. For example, in some embodiments, the polynucleotide as described herein may comprise one or more heavy chain complementarity-determining regions (HCDRs) (e.g., HCDR1, HCDR2, and / or HCDR3) from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotide as described herein may comprise HCDR1, HCDR2, and HCDR3 from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotide as described herein may comprise a heavy chain variable domain from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotide as described herein may comprise one or more light chain complementarity-determining regions (LCDRs) (e.g., LCDR1, LCDR2, and / or LCDR3) from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotides described herein may comprise LCDR1, LCDR2, and LCDR3 from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400. In some embodiments, the polynucleotides described herein may comprise a light chain variable domain from 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400.

[0430] In some embodiments, multiple polynucleotides of the immunoglobulin chains each encoding an antibody agent can be used to deliver (e.g., by administration to a subject) two or more antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400, or fragments or variants thereof). In some embodiments, multiple polynucleotides of the immunoglobulin chains each encoding an antibody agent can be used to deliver (e.g., by administration to a subject) three or more antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400, or fragments or variants thereof). In some embodiments, multiple polynucleotides of immunoglobulin chains, each encoding an antibody agent, can be used to deliver (e.g., by administration to a subject) four or more antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400, or fragments or variants thereof). In some embodiments, multiple polynucleotides of immunoglobulin chains, each encoding an antibody agent, can be used to deliver (e.g., by administration to a subject) two, three, four, five, or six antibody agents (e.g., 1-18, PGT121, 3BNC117, b12, 10-1074, 10E8, 10E8v4, VRC01, VRC07-523-L / S, or PGDM1400, or fragments or variants thereof). In some embodiments, the antibody agent encoded by one or more polynucleotides described herein includes all or part of the PGDM1400 antibody.

[0431] Antibody agents as described herein (e.g., PGDM1400 antibody agent) may be characterized by the amino acid sequence of one or more domains within their antibody structure. For example, the antibody agent may comprise, for example, at least one heavy (H) chain and at least one light (L) chain linked by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated as VL herein) and a light chain constant region. The variable regions of each light chain / heavy chain (VL / VH) pair form an antigen-binding domain.

[0432] Within each of the light or heavy chain variable domains, there are three short segments, referred to as complementarity-determining regions (“CDRs”). The six CDRs in the antibody variable domain (three in the light chain variable domain and three in the heavy chain variable domain) fold together in three-dimensional space to form the actual antibody binding site. As used herein, the terms “LCDR1,” “LCDR2,” and “LCDR3” refer to CDRs 1, 2, and 3 of the variable light (L) chain of the antibody agent. In some embodiments, the light chain variable domains provided herein include LCDR1, LCDR2, and LCDR3 in the N-terminal to C-terminal direction. Similarly, as used herein, the terms “HCDR1,” “HCDR2,” and “HCDR3” refer to CDRs 1, 2, and 3 of the variable heavy (H) chain of the antibody agent. In some embodiments, the heavy chain variable domains provided herein include HCDR1, HCDR2, and HCDR3 in the N-terminal to C-terminal direction.

[0433] The regions within the variable region but not within the CDR are called frame regions (“FRs”). Therefore, frame regions are scattered among the complementarity-determining regions (CDRs). Thus, each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Generally, in naturally occurring antibodies, frame regions are more conserved than variable regions.

[0434] The PGDM1400 antibody (including its nucleotide and amino acid sequences) is disclosed in WO2021 / 087015 and U.S. Patent No. 10,093,720, which are incorporated herein by reference in their entirety.

[0435] In some embodiments, the PGDM1400 antibody comprises HCDR1, HCDR2, and HCDR3 having a heavy chain variable domain having the amino acid sequence SEQ ID NO:36. In some embodiments, the PGDM1400 antibody comprises LCDR1, LCDR2, and LCDR3 having a light chain variable domain having the amino acid sequence SEQ ID NO:43.

[0436] The locations of CDRs and frame regions within the VH and VL domains of the antibody agents described herein (e.g., PGDM1400 antibody agent) can be determined using various numbering systems known in the art, such as Kabat, Chothia, AbM, and IMGT (see, for example, Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J. Mol. Biol., 273:927-748 (1997) ImMunoGenTics (IMGT) numbering; Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), each of which is incorporated herein by reference. Therefore, CDRs within the same VH or VL domain in the PGDM1400 antibody can be determined using different numbering systems.

[0437] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3, representing heavy chain variable domains, as shown in SEQ ID NO:36, according to Kabat numbering. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3, representing heavy chain variable domains, as shown in SEQ ID NO:36, according to Chothia numbering. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3, representing heavy chain variable domains, as shown in SEQ ID NO:36, according to IMGT numbering. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of HCDR1, HCDR2, and HCDR3, representing heavy chain variable domains, as shown in SEQ ID NO:36, according to AbM numbering.

[0438] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3, which are represented by the light chain variable domains: amino acid sequence SEQ ID NO:43, according to Kabat number. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3, which are represented by the light chain variable domains: amino acid sequence SEQ ID NO:43, according to Chothia number. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3, which are represented by the light chain variable domains: amino acid sequence SEQ ID NO:43, according to IMGT number. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3, which are represented by the light chain variable domains: amino acid sequence SEQ ID NO:43, according to AbM number.

[0439] In some embodiments, the HCDR1 of the antibody (e.g., PGDM1400 antibody) contains an amino acid sequence as shown in SEQ ID NO:18, 1484, or 1490.

[0440] In some embodiments, the HCDR2 of the antibody (e.g., PGDM1400 antibody) contains an amino acid sequence as shown in SEQ ID NO:21, 1485, 1491 or 1492.

[0441] In some embodiments, the HCDR3 of the antibody (e.g., PGDM1400 antibody) contains an amino acid sequence as shown in SEQ ID NO:24, 1486, or 1493.

[0442] In some embodiments, the LCDR1 of the antibody (e.g., PGDM1400 antibody) contains an amino acid sequence as shown in SEQ ID NO:27 or 1487.

[0443] In some embodiments, the LCDR2 of the antibody (e.g., PGDM1400 antibody) contains an amino acid sequence as shown in SEQ ID NO:30 or 1488.

[0444] In some embodiments, the LCDR3 of the antibody (e.g., PGDM1400 antibody) contains an amino acid sequence as shown in SEQ ID NO:33 or 1489.

[0445] In some implementations, the antibody agent (e.g., PGDM1400 antibody agent) contains one or more CDRs or any set of CDRs, as shown in Table 2 below.

[0446] Table 2: Exemplary CDR sequences of PGDM1400 antibody

[0447]

[0448]

[0449] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO: 1484, 1492, and 1486, respectively. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO: 1490, 1491, and 1486, respectively. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO: 18, 21, and 1493, respectively.

[0450] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:18, 21, and 24, respectively. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:1484, 1485, and 1486, respectively.

[0451] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO: 1487, 1488, and 1489, respectively. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO: 27, 30, and 1489, respectively.

[0452] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:27, 30, and 33, respectively.

[0453] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:18, 21, and 24, respectively; and (ii) the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:27, 30, and 33, respectively.

[0454] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:1484, 1485, and 1486, respectively; and (ii) the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:1487, 1488, and 1489, respectively.

[0455] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:1490, 1491, and 1486, respectively; and (ii) the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:1487, 1488, and 1489, respectively.

[0456] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:1484, 1492, and 1486, respectively; and (ii) the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:1487, 1488, and 1489, respectively.

[0457] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:1490, 1491, and 1486, respectively; and (ii) the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:1487, 1488, and 1489, respectively.

[0458] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) the amino acid sequences HCDR1, HCDR2, and HCDR3, as shown in SEQ ID NO:18, 21, and 1493, respectively; and (ii) the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO:27, 30, and 1489, respectively.

[0459] In some embodiments, the PGDM1400 antibody agent comprises FR1, FR2, FR3, and / or FR4 domains of the heavy chain variable domain, represented by the amino acid sequence SEQ ID NO:36. In some embodiments, the PGDM1400 antibody agent comprises FR1, FR2, FR3, and / or FR4 domains of the light chain variable domain, represented by the amino acid sequence SEQ ID NO:43.

[0460] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a heavy chain FR1, which contains the amino acid sequence shown in SEQ ID NO:1496. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a heavy chain FR2, which contains the amino acid sequence shown in SEQ ID NO:1497. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a heavy chain FR3, which contains the amino acid sequence shown in SEQ ID NO:1498. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a heavy chain FR4, which contains the amino acid sequence shown in SEQ ID NO:1499.

[0461] In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a light chain FR1, which contains the amino acid sequence shown in SEQ ID NO:1500. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a light chain FR2, which contains the amino acid sequence shown in SEQ ID NO:1501. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a light chain FR3, which contains the amino acid sequence shown in SEQ ID NO:1502. In some embodiments, the antibody agent (e.g., PGDM1400 antibody agent) comprises a light chain FR4, which contains the amino acid sequence shown in SEQ ID NO:1503.

[0462] In some embodiments, the heavy chain FR1, FR2, FR3, and / or FR4 domains of the antibody agent (e.g., PGDM1400 antibody agent) comprise amino acid sequences as shown in SEQ ID NO: 1496, 1497, 1498, and 1499, respectively. In some embodiments, the light chain FR1, FR2, FR3, and / or FR4 domains of the antibody agent (e.g., PGDM1400 antibody agent) comprise amino acid sequences as shown in SEQ ID NO: 1500, 1501, 1502, and 1503, respectively.

[0463] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises a VH domain containing the amino acid sequences HCDR1, FR1, HCDR2, and HCDR3, as shown in SEQ ID NO: 18, 1496, 21, and 24, respectively.

[0464] In some embodiments, the antibody (e.g., PGDM1400 antibody) comprises (i) a VH domain containing the amino acid sequences HCDR1, FR1, HCDR2, and HCDR3, as shown in SEQ ID NO: 18, 1496, 21, and 24, respectively; and (ii) a VL domain containing the amino acid sequences LCDR1, LCDR2, and LCDR3, as shown in SEQ ID NO: 27, 30, and 33, respectively.

[0465] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein comprises all or part of the PGDM1400 antibody. In some embodiments, the antibody agent comprises a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof. In some embodiments, the antibody agent comprises a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24). In some embodiments, the antibody agent comprises a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof. In some embodiments, the antibody agent comprises a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33). In some embodiments, the antibody agent comprises (a) a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof; and (b) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof. In some embodiments, the antibody agent comprises (a) a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24); and (b) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33).

[0466] In some embodiments, the polynucleotides described herein encode all or part of the PGDM1400 antibody. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO:18); (ii) HCDR2 (SEQ ID NO:21); (iii) HCDR3 (SEQ ID NO:24); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO:18); (ii) HCDR2 (SEQ ID NO:21); and (iii) HCDR3 (SEQ ID NO:24). In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a light chain variable domain, wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); (iii) LCDR3 (SEQ ID NO:33); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof; and the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof.In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24); and the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33). In some embodiments, the polynucleotides described herein encode two immunoglobulin chains: a first immunoglobulin chain comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof; and a second immunoglobulin chain comprising a light chain variable domain, wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode two immunoglobulin chains: a first immunoglobulin chain comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24); and a second immunoglobulin chain comprising a light chain variable domain, wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33).

[0467] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein includes a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:36. In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein includes a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, the antibody agent includes the heavy chain variable domain represented by SEQ ID NO:36. In some embodiments, the antibody agent includes the light chain variable domain represented by SEQ ID NO:43.

[0468] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., PGDM1400 antibody agent) comprises a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:1494. In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., PGDM1400 antibody agent) comprises a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, the antibody agent comprises the heavy chain variable domain represented by SEQ ID NO:1494. In some implementations, the antibody agent comprises the light chain variable domain represented by SEQ ID NO:43.

[0469] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., the PGDM1400 antibody agent) comprises a heavy chain variable domain containing one or more mutations relative to the amino acid sequence represented by SEQ ID NO:1494. In some embodiments, a substitution mutation is present at residue 3 and / or 5 relative to SEQ ID NO:1494. In some embodiments, the substitution mutation at residue 3 results in a positively charged amino acid present at residue 3 relative to SEQ ID NO:1494. Positively charged amino acids include Lys(K), Arg(R), and His(H). In some embodiments, the substitution mutation at residue 3 relative to SEQ ID NO:1494 comprises Q3H. In some embodiments, the substitution mutation at residue 5 results in a polar amino acid present at residue 5 relative to SEQ ID NO:1494. Polar amino acids include Ser(S), Thr(T), Tyr(Y), Asn(N), and Gln(Q). In some embodiments, the substitution mutation at residue 5 relative to SEQ ID NO:1494 comprises V5T.

[0470] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., the PGDM1400 antibody agent) includes a heavy chain variable domain containing SEQ ID NO:1494, having substitution mutations Q3H and V5T.

[0471] In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain, said heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:36. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a light chain variable domain, said light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, the polynucleotides described herein encode an immunoglobulin chain comprising a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:36, and wherein the light chain variable domain has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:43.

[0472] In some embodiments, the polynucleotides as described herein encode an immunoglobulin chain of the antibody agent, wherein the immunoglobulin chain comprises a heavy chain variable (VH) domain. In some embodiments, the VH domain comprises the VH domain of the PGDM1400 antibody. In some embodiments, the polynucleotides encode a VH domain of an antibody selected from: PGT121, 3BNC117, b12, 10-1074, 10-1074-LS, 10E8, VRC01, VRC07-523, or 1-18 (e.g., as described herein).

[0473] In some embodiments, the polynucleotide comprises a VH domain coding sequence comprising (a) an HCDR1 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:19; (b) an HCDR2 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:22; (c) an HCDR3 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:25; or (d) a combination thereof. In some embodiments, the polynucleotide comprises a VH domain coding sequence comprising (a) an HCDR1 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:19; (b) an HCDR2 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:22; and (c) an HCDR3 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:25. In some embodiments, the polynucleotide encodes a VH domain and comprises a VH coding sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that in SEQ ID NO: 37, 39, or 41. In some embodiments, the polynucleotide encodes a VH domain and comprises a VH coding sequence according to SEQ ID NO: 37, 39, or 41.

[0474] In some embodiments, the polynucleotide as described herein comprises an immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a light chain variable (VL) domain. In some embodiments, the VL domain comprises the VL domain of the PGDM1400 antibody. In some embodiments, the polynucleotide encodes the VL domain of an antibody selected from: PGT121, 3BNC117, b12, 10-1074, 10-1074-LS, 10E8, VRC01, VRC07-523, or 1-18 (e.g., as described herein).

[0475] In some embodiments, the polynucleotide comprises a coding region of one or more immunoglobulin chains encoding an antibody agent, wherein the immunoglobulin chains comprise a light chain variable (VL) domain. In some embodiments, the polynucleotide comprises a VL domain coding sequence comprising (a) an LCDR1 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:28; (b) an LCDR2 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:31; (c) an LCDR3 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:34; or (d) a combination thereof. In some embodiments, the polynucleotide comprises a VL domain coding sequence comprising (a) an LCDR1 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:28; (b) an LCDR2 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:31; and (c) an LCDR3 coding sequence comprising a ribonucleic acid sequence according to SEQ ID NO:34. In some embodiments, the polynucleotide encodes a VL domain and comprises a VL coding sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that in SEQ ID NO:44 or 46. In some embodiments, the polynucleotide encodes a VL domain and comprises a VL coding sequence according to SEQ ID NO:44 or 46.

[0476] In some implementations, the antibody agent is formed from one, two, three, or four immunoglobulin chains.

[0477] In some embodiments, the polynucleotides as described herein encode a single immunoglobulin chain. In some embodiments, a first polynucleotide encodes a first immunoglobulin chain of the antibody agent. In some embodiments, a first polynucleotide encodes a first immunoglobulin chain of the antibody agent and a second polynucleotide encodes a second immunoglobulin chain of the antibody agent. In some embodiments, a first polynucleotide encodes a first immunoglobulin chain of the antibody agent, a second polynucleotide encodes a second immunoglobulin chain of the antibody agent, and a third polynucleotide encodes a third immunoglobulin chain of the antibody agent. In some embodiments, a first polynucleotide encodes a first immunoglobulin chain of the antibody agent, a second polynucleotide encodes a second immunoglobulin chain of the antibody agent, a third polynucleotide encodes a third immunoglobulin chain of the antibody agent, and a fourth polynucleotide encodes a fourth immunoglobulin chain of the antibody agent.

[0478] In some embodiments, the polynucleotide as described herein encodes two immunoglobulin chains. In some embodiments, a single polynucleotide may include a first coding region encoding a first immunoglobulin chain of an antibody and a second coding region encoding a second immunoglobulin chain of an antibody. In some embodiments, the first and second coding regions are separated by an internal ribosome entry side (IRES), an internal promoter, or a peptide sequence (e.g., a “self-cleaving” 2A or 2A-like sequence) (see, for example, Szymczak et al., Nat Biotechnol 22:589, May 2004; ePub, April 4, 2004, which is incorporated herein by reference) to generate the first and second immunoglobulin chains from a single polynucleotide.

[0479] Antibody agents encoded by one or more polynucleotides described herein can be in the various forms described herein. Exemplary types of antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, the antibody agent may include one or more humanized, chimeric, or other sequence elements, as known in the art. In some embodiments, the antibody agent used according to this disclosure is selected from, but is not limited to, intact IgG, IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., ...). (etc.); CrossMab (e.g., CrossMab) CH1-CLx CrossMab CH1-CLcv CrossMab with bispecificity of pestle and mortar CH1-CLx Antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining regions (CDRs) or sets thereof; single-chain Fv (scFv); scFv-Fc fusions; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel-shaped antibodies; masked antibodies (e.g., Small modular immunotherapies (“SMIPs™”); single-chain or tandem bifunctional antibodies VHH; Small antibodies; Ankylosing spondylogenetics or DART; TCR-like antibodies; MicroProteins; as well as In some implementations, immunoglobulin chains and / or fragments of such antibodies can be used in combination, for example, the scFv-Fc arm can be used in combination with a conventional antibody arm.

[0480] The following describes further exemplary forms that can be used according to this disclosure.

[0481] A. Conventional antibodies

[0482] In some implementations, the polynucleotides described herein can be used to express conventional antibodies. As used herein, "conventional antibody" refers to an antibody agent comprising two heavy chains and two light chains (see, for example...). Figure 5 (Figure A). Each heavy chain includes a heavy chain variable structural domain operatively connected to one or more heavy chain constant structural domains. In some embodiments, one or more heavy chain constant structural domains comprise a CH1 structural domain, a hinge structural domain, a CH2 structural domain, a CH3 structural domain, or a combination thereof. In some cases, one or more heavy chain constant structural domains comprise a CH1 structural domain, a hinge structural domain, a CH2 structural domain, a CH3 structural domain, a CH4 structural domain, or a combination thereof. Each light chain includes a light chain variable structural domain operatively connected to a light chain constant structural domain.

[0483] Typically, both heavy-chain and light-chain variable domains can be further subdivided into variant regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each of these heavy-chain and light-chain variable domains may include, for example, three CDRs and four framework regions, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, one or more of which can be engineered as described herein. The CDRs in the heavy chain are designated as "HCDR1", "HCDR2", and "HCDR3", respectively, and the CDRs in the light chain are designated as "LCDR1", "LCDR2", and "LCDR3", respectively.

[0484] The conventional antibodies described herein may comprise any of the five major antibody classes: IgA, IgD, IgE, IgG, and IgM. In some embodiments, the conventional antibody comprises an IgG or IgA antibody. In some embodiments, the conventional antibody described herein comprises a specific isotype selected from the IgA and IgG isotype groups: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Furthermore, in some embodiments, the conventional antibody may comprise any specific heavy chain constant domain, which corresponds to different classes of immunoglobulins, including α, δ, ε, γ, and μ. In some embodiments, the conventional antibody is a complete IgG1 antibody or other antibody classes or isotypes as described herein. (See, for example, Hudson et al., Nat. Med., 9:129-134 (2003); Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, pp. 269-315 (1994); Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); WO93 / 01161; and U.S. Patent Nos. 5,571,894, 5,869,046, 6,248,516 and 5,587,458, each of which is incorporated herein by reference). In addition to the various isotypes, allelic variations exist among IgG subclasses, resulting in allotypes or allotropes. The IgG antibody agents described herein may contain specific allotypes, including but not limited to G1m3, G1m17, G1m17,1 or G1m17,1,2 or G1m3,1 (see Vidarsson et al., Front. Immunol, 5(520):1-17, 2014, which is incorporated herein by reference in its entirety).

[0485] The Fc region of a conventional antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells mediating cytotoxicity. As is known in the art, the affinity and / or other binding properties of the Fc region to Fc receptors can be modulated by glycosylation or other modifications. In some embodiments, conventional antibodies generated and / or utilized according to the invention include glycosylated Fc domains, including modified or engineered such glycosylated Fc domains. In some embodiments, the conventional antibody is naturally occurring (e.g., generated by an organism reacting with an antigen) or generated by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, the conventional antibody is polyclonal; in some embodiments, the conventional antibody is monoclonal. In some embodiments, the conventional antibody has a constant region sequence characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the conventional antibody sequence elements are humanized, primate-derived, chimeric, etc., as known in the art.

[0486] Conventional antibodies, as described herein, are those with a structure generally similar to that of native antibodies or those with a heavy chain containing an Fc region as defined herein.

[0487] In some embodiments, conventional antibodies encoded by one or more polynucleotides provided herein comprise all or part of the PGDM1400 antibody. In some embodiments, the conventional antibody comprises a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof. In some embodiments, the conventional antibody comprises a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24). In some embodiments, the conventional antibody comprises a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof. In some implementations, the conventional antibody includes a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); and (iii) LCDR3 (SEQ ID NO:33). In some embodiments, the conventional antibody comprises (a) a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof; and (b) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof. In some embodiments, the conventional antibody comprises (a) a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24); and (b) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24); and (iv) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 28 ... (iii) HCDR3 (SEQ ID NO: 24); and (iv) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 28); (ii) LCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); and (iv) a light chain variable domain comprising: (i) LCDR1 (SEQ ID (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33).

[0488] In some embodiments, the polynucleotides described herein encode immunoglobulin chains of conventional antibodies, wherein the immunoglobulin chains comprise heavy chain variable domains, and wherein the heavy chain variable domains comprise: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode immunoglobulin chains of conventional antibodies, wherein the immunoglobulin chains comprise heavy chain variable domains, and wherein the heavy chain variable domains comprise: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24). In some embodiments, the polynucleotides described herein encode immunoglobulin chains of conventional antibodies, wherein the immunoglobulin chains comprise light chain variable domains, and wherein the light chain variable domains comprise: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); (iii) LCDR3 (SEQ ID NO:33); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode immunoglobulin chains of conventional antibodies, wherein the immunoglobulin chains comprise light chain variable domains, and wherein the light chain variable domains comprise: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); and (iii) LCDR3 (SEQ ID NO:33). In some embodiments, the polynucleotides described herein encode two immunoglobulin chains of a conventional antibody: a first immunoglobulin chain comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof; and a second immunoglobulin chain comprising a light chain variable domain, wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) combinations thereof.In some embodiments, the polynucleotides described herein encode two immunoglobulin chains of a conventional antibody: a first immunoglobulin chain comprising a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24); and a second immunoglobulin chain comprising a light chain variable domain, wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33).

[0489] In some embodiments, the conventional antibody encoded by one or more polynucleotides provided herein includes a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:36. In some embodiments, the conventional antibody encoded by one or more polynucleotides provided herein includes a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, the conventional antibody includes the heavy chain variable domain represented by SEQ ID NO:36. In some embodiments, the conventional antibody includes the light chain variable domain represented by SEQ ID NO:43.

[0490] In some embodiments, the conventional antibody encoded by one or more polynucleotides provided herein includes a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:1494. In some embodiments, the conventional antibody encoded by one or more polynucleotides provided herein includes a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, the conventional antibody includes the heavy chain variable domain represented by SEQ ID NO:1494. In some embodiments, the conventional antibody includes the light chain variable domain represented by SEQ ID NO:43.

[0491] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., the PGDM1400 antibody agent) comprises a heavy chain variable domain containing one or more mutations relative to the amino acid sequence represented by SEQ ID NO:1494. In some embodiments, a substitution mutation is present at residue 3 and / or 5 relative to SEQ ID NO:1494. In some embodiments, the substitution mutation at residue 3 results in a positively charged amino acid present at residue 3 relative to SEQ ID NO:1494. Positively charged amino acids include Lys(K), Arg(R), and His(H). In some embodiments, the substitution mutation at residue 3 relative to SEQ ID NO:1494 comprises Q3H. In some embodiments, the substitution mutation at residue 5 results in a polar amino acid present at residue 5 relative to SEQ ID NO:1494. Polar amino acids include Ser(S), Thr(T), Tyr(Y), Asn(N), and Gln(Q). In some embodiments, the substitution mutation at residue 5 relative to SEQ ID NO:1494 comprises V5T.

[0492] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., the PGDM1400 antibody agent) includes a heavy chain variable domain containing SEQ ID NO:1494, having substitution mutations Q3H and V5T.

[0493] In some embodiments, the polynucleotides described herein encode immunoglobulin chains of conventional antibodies, wherein the immunoglobulin chains include heavy chain variable domains having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:36. In some embodiments, the polynucleotides described herein encode immunoglobulin chains of conventional antibodies, wherein the immunoglobulin chains include light chain variable domains having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:43.

[0494] Conventional antibodies encoded by one or more polynucleotides as described herein may comprise one or more heavy chain constant domains. In some embodiments, the one or more heavy chain constant domains comprise a CH3 domain. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein comprises a CH3 domain, said CH3 domain comprising an allotype of G1m3, G1m17, or G1m17,1. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein comprises a CH3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence represented by any one of SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, or 1495. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein includes a CH3 domain having an amino acid sequence represented by any one of SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, or 1495. In some embodiments, the CH3 domain includes the amino acid sequence SEQ ID NO: 1495, having the substitution mutations D16E and L18M.

[0495] Conventional antibodies encoded by one or more polynucleotides as described herein may comprise one or more heavy chain constant domains containing amino acid modifications (e.g., substitutions or deletions) at one or more amino acid positions. For example, a conventional antibody encoded by one or more polynucleotides as described herein may include an L / S mutation in the CH3 region (for enhanced FcRn binding) (see Zalevsky J et al., Nat Biotechnol. 2010, which is incorporated herein by reference). Such mutations are labeled M428L and N434S according to EU numbers (i.e., M88L and N94S within the CH3 domain, e.g., SEQ ID NO: 113, 116, or 1495) and are referred to herein as “LS” or “L / S” (see, e.g.) Figure 5 (Figure D). In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains an E294 deletion (for Fc hypersialylation) (see Bas M et al. J Immunol 2019, which is incorporated herein by reference).

[0496] This disclosure also provides techniques that can be used to express antibody agents, such as... Figure 3As shown or described below: Stadler et al. (2016) Oncoimmunology 5(3):e1091555; and / or Stadler et al. (2017) Nature Medicine 23(7):815-817. Generating multiple antibody agents from a single composition (e.g., a composition containing enough polynucleotides to encode multiple antibody agents) is challenging, especially because random pairing of different antibody heavy and light chains can produce unwanted antibody species. In these cases, complex purification procedures are required to isolate the desired antibody agent due to the presence of mismatch byproducts and significantly reduced yields (see, for example, Morrison, SL, Nature Biotech. 25, 1233-1234, 2007, which is incorporated herein by reference). Generally, the problem of mismatch byproducts persists even when recombinant expression techniques are used. One approach to addressing the mismatch byproduct problem is known as the "mortar and pestle technique" (KIH), which aims to modify the contact interface by introducing a mutation in the CH3 domain, thereby forcing two different antibody heavy chains to pair. On one chain, a large amino acid is replaced by an amino acid with a short side chain to form a "mortar," and an amino acid with a large side chain is introduced into another CH3 domain to form a "pestle." By co-expressing these two heavy chains and two light chains, a higher yield of heterodimers was observed than that of homodimers (see Ridgway, JB et al., Protein Eng. 9, 617-621, 1996; and WO 96 / 027011, which is incorporated herein by reference). In some embodiments, the antibody agents described herein utilize KIH technology, as described, for example, in WO 1998 / 050431, which is incorporated herein by reference in its entirety. As described herein, the antibody agents may contain certain mutations utilizing KIH technology, including but not limited to CH3 modifications. In some embodiments, the antibody agents contain a CH3 domain containing one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU numbers). In some embodiments, the antibody agent comprises a CH3 domain, wherein the CH3 domain comprises each of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU designations). Such combinations of mutations are referred to herein as "cah". In some embodiments, the antibody agent comprises a CH3 domain, wherein the CH3 domain comprises one or more mutations selected from the following: S354C and T366W (according to EU designations). In some embodiments, the antibody agent comprises a CH3 domain, wherein the CH3 domain comprises each of the following mutations: S354C and T366W (according to EU designations). Such combinations of CH3 mutations are referred to herein as "cak".

[0497] Therefore, in some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein include a CH3 domain, said CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU designations). In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein include a CH3 domain, said CH3 domain comprising one or more mutations selected from: S354C and T366W (according to EU designations).

[0498] In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: M428L, N434S, or a combination thereof (e.g., an "L / S" mutation). In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence containing any of SEQ ID NO: 117 and 135. In some embodiments, the polynucleotide encodes a CH3 domain comprising one or more of the following substitution mutations: Y349C, T366S, L368A, and Y407V (according to EU numbers). In some embodiments, the polynucleotide comprises ribonucleic acid sequences according to SEQ ID NO: 120 and 138. In some embodiments, the polynucleotide encodes a CH3 domain comprising one or both of the following substitution mutations: S354C and T366W (according to EU numbers). In some embodiments, the polynucleotide comprises ribonucleic acid sequences according to SEQ ID NO: 126 and 144.

[0499] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operatively linked to one or more constant domains, wherein the one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NO: 114. In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence encoding a CH3 domain comprising an allotype of G1m3, G1m17, or G1m17,1. In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence containing either SEQ ID NO: 114 or 132.

[0500] In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein includes a CH1 domain. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein includes a CH1 domain containing the G1m3 allotype. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein includes a CH1 domain containing the G1m17 allotype. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein includes a CH1 domain containing the amino acid sequence represented by SEQ ID NO: 76 or 81.

[0501] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operatively linked to one or more constant domains, wherein the one or more constant domains comprise a CH1 domain. In some embodiments, the polynucleotide comprises a CH1 ribonucleic acid sequence according to SEQ ID NO: 77 or 79. In some embodiments, the polynucleotide encodes a CH1 domain comprising a G1m3 allotype. In some embodiments, the polynucleotide encodes a CH1 domain comprising a G1m17 allotype. In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence according to SEQ ID NO: 77, 79, or 82.

[0502] In some embodiments, the polynucleotide encodes a CH1 domain containing one or more mutations. In some embodiments, the polynucleotide encodes a CH1 domain containing one or more added serine residues. In some embodiments, the polynucleotide encodes a CH1 domain containing two additional added serine residues (referred to herein as "SS"). In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 91 or 94. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence represented by SEQ ID NO: 91 or 94. In some embodiments, the polynucleotide encodes a CH1 domain containing one or more charge-variant mutations. In some embodiments, the polynucleotide encodes a CH1 domain comprising one or more substitution mutations selected from K147E, K213D, or combinations thereof. In some embodiments, the polynucleotide encodes a CH1 ribonucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 85 or 88. In some embodiments, the polynucleotide encodes the CH1 ribonucleic acid sequence according to SEQ ID NO: 85 or 88.

[0503] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a hinge domain. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a hinge domain containing the amino acid sequence represented by SEQ ID NO:161 (referred to herein as the “hinge” in Tables 2 and 4).

[0504] In some embodiments, the polynucleotide encodes a hinge domain. In some embodiments, the polynucleotide encodes the hinge ribonucleic acid sequence represented by SEQ ID NO:162. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification comprising the deletion of one or more amino acid residues. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification comprising the deletion of the amino acid residue EPKSC in a conventional Ig hinge domain (represented by SEQ ID NO:161). Such modifications are referred to herein as “hinge_del” or “ΔEPKSC”. In some embodiments, the polynucleotide encodes the hinge ribonucleic acid sequence represented by SEQ ID NO:168. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification comprising a C220S mutation (according to EU designation). Such mutant hinge domains are referred to herein as “hinge_S” or “C / S”. In some implementations, the polynucleotide encodes the hinged ribonucleic acid sequence represented by SEQ ID NO:165.

[0505] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a CH2 domain. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that represented by SEQ ID NO:96. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a CH2 domain having an amino acid sequence represented by SEQ ID NO:96.

[0506] In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein include a CH2 domain having one or more mutations (e.g., relative to SEQ ID NO:96). For example, in some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein include one or more of the following mutations: G236A, A330L, and I332E (according to EU designations). In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein include the following mutations: G236A, A330L, and I332E (according to EU designations), referred to herein as “GAALIE”. Such mutations in the CH2 domain are associated with increased affinity for Fc receptors FcgRIIA and FcgRIII, thereby enhancing antibody effector function.

[0507] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 99 or 102. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a CH2 domain having an amino acid sequence represented by SEQ ID NO: 99 or 102.

[0508] In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains one or more mutations selected from G236A and I332E (according to EU designations). In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains mutations selected from G236A and I332E (according to EU designations), referred to herein as “GAIE”. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO:104. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a CH2 domain having the amino acid sequence represented by SEQ ID NO:104.

[0509] In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a mutation: G236A (according to EU designation), referred to herein as "GA". In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO:107. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a CH2 domain having the amino acid sequence represented by SEQ ID NO:107.

[0510] In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a mutation: I332E (according to EU designation), referred to herein as "IE". In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a CH2 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO:110. In some embodiments, the conventional antibody encoded by one or more polynucleotides as described herein contains a CH2 domain having an amino acid sequence represented by SEQ ID NO:110.

[0511] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operatively linked to one or more constant domains, wherein the one or more constant domains comprise a CH2 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NO:97. In some embodiments, the CH2 ribonucleic acid encodes a CH2 domain having one or more amino acid substitution mutations. For example, in some embodiments, the CH2 ribonucleic acid sequence encodes one or more of the following mutations: G236A, A330L, and I332E (according to EU designation), referred to herein as “GAALIE”. Such mutations in the CH2 domain are associated with increased affinity for Fc receptors FcgRIIA and FcgRIII, thereby enhancing antibody effector function. In some embodiments, the CH2 ribonucleic acid sequence comprises or consists of a sequence according to SEQ ID NO:100 or 103. In some embodiments, the CH2 ribonucleic acid sequence encodes one or more of the following mutations: G236A and I332E (according to EU designation), referred to herein as “GAIE”. In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO:105. In some embodiments, the CH2 ribonucleic acid sequence encodes the mutation G236A (according to EU designation), referred to herein as “GA”. In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO:108. In some embodiments, the CH2 ribonucleic acid sequence encodes the mutation I332E (according to EU designation), referred to herein as “IE”. In some embodiments, the CH2 ribonucleic acid sequence comprises the sequence according to SEQ ID NO:111. In some embodiments, the CH2 ribonucleic acid sequence encodes the CH2 domain, which includes an E294 deletion (according to EU designation).

[0512] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a signal peptide, said signal peptide including a human signal peptide. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a signal peptide, said signal peptide including SEQ ID NO:1.

[0513] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a light chain constant domain, wherein the light chain constant domain includes a κ light chain constant domain. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a κ light chain constant domain having an amino acid sequence that is at least 80, 85, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO:149. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a κ light chain constant domain having the amino acid sequence represented by SEQ ID NO:149. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein includes a λ chain variable domain.

[0514] In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein comprise immunoglobulin chains (e.g., immunoglobulin heavy chains) encoded by nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequences represented by SEQ ID NO: 170-265, 1305-1306, 1308-1309, and 1314-1315. In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein comprise immunoglobulin chains (e.g., immunoglobulin heavy chains) encoded by nucleic acid sequences represented by any one of SEQ ID NO: 170-265, 1305-1306, 1308-1309, and 1314-1315. In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein comprise immunoglobulin chains (e.g., immunoglobulin light chains) encoded by nucleic acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:842, 843, and 1311-1323. In some embodiments, conventional antibodies encoded by one or more polynucleotides as described herein comprise immunoglobulin chains (e.g., immunoglobulin light chains) encoded by nucleic acid sequences represented by SEQ ID NO:842-843 and 1311-1312.

[0515] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein comprises an immunoglobulin chain (e.g., an immunoglobulin heavy chain) containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence represented by any one of SEQ ID NO: 1307, 1310, or 1316. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein comprises an immunoglobulin chain (e.g., an immunoglobulin heavy chain) containing an amino acid sequence represented by any one of SEQ ID NO: 1307, 1310, and 1316.

[0516] In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) containing an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1313. In some embodiments, a conventional antibody encoded by one or more polynucleotides as described herein comprises an immunoglobulin chain (e.g., an immunoglobulin light chain) containing the amino acid sequence represented by SEQ ID NO:1313.

[0517] Exemplary immunoglobulin chain (e.g., immunoglobulin heavy or light chain) configurations of conventional antibodies as described herein are shown in Table 3 below.

[0518] Table 3: Exemplary Immunoglobulin Chain Configurations

[0519]

[0520]

[0521]

[0522]

[0523] B.CrossMab CH1-CLx

[0524] This disclosure also provides techniques for delivering and expressing antibody agents as described herein in the form of “CrossMab” (see, for example, WO2015 / 101588A1, WO 2009 / 080253A1, and Schaefer, W. et al., PNAS, 108, 11187-1191, 2011, which are incorporated herein by reference in their entirety). In some embodiments, the antibody agent in the form of CrossMab contains a CL-CH1 cross (referred to herein as “Cross Mab”) in one or both binding arms. CH1-CLx (or "CH1-CLx"). Such modifications reduce the formation of byproducts caused by erroneous heavy chain mismatches between the light chain of the first antibody that specifically binds to the first antigen and the heavy chain of the second antibody that specifically binds to the second antigen (when compared to methods without such domain exchanges).

[0525] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein comprises a first immunoglobulin chain and a second immunoglobulin chain. In some embodiments, the polynucleotide may encode CrossMab as described herein. CH1-CLx The antibody agent comprises a first immunoglobulin chain and a second immunoglobulin chain. In some embodiments, the polynucleotide encoding the first immunoglobulin chain comprises a ribonucleic acid sequence encoding a VH domain, a CL domain, a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the polynucleotide encoding the second immunoglobulin chain comprises a ribonucleic acid sequence encoding a light chain variable (VL) domain and a CH1 domain (see, for example...). Figure 4 (Figure B). In some implementations, CrossMab is encoded. CH1-CLx The antibody agent contains a polynucleotide sequence encoding any of the immunoglobulin chain configurations in Table 4, corresponding to SEQ ID NO: 266-361, 1317-1318, 1329-1330, 1332-1333, 1335-1336, and 1338-1339. In some embodiments, it encodes CrossMab. CH1-CLx The antibody polynucleotide contains a ribonucleic acid sequence encoding any of the immunoglobulin chain configurations in Table 4, corresponding to SEQ ID NO: 844-847 and 1230-1321.

[0526] In some implementations, CrossMab CH1-CLx The antibody agent may be encoded by two separate polynucleotides: a first polynucleotide containing a coding region that encodes (in 5' to 3' order): a heavy chain variable domain (VH), a light chain constant domain (CL), a hinge region, a CH2 domain, and a CH3 domain (see example...). Figure 9(Figure A); and a second polynucleotide comprising a coding region encoding (in 5' to 3' order): a light chain variable domain (VL) and a CH1 domain (see, for example) Figure 9 (Figure B).

[0527] In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent includes all or part of the PGDM1400 antibody. In some embodiments, CrossMab... CH1-CLx The antibody agent comprises a heavy chain variable domain, said heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof. In some embodiments, CrossMab CH1-CLx The antibody agent comprises a heavy chain variable domain, said heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24). In some embodiments, CrossMab CH1-CLx The antibody agent comprises a light chain variable domain, said light chain variable domain comprising: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); (iii) LCDR3 (SEQ ID NO:33); or (iv) combinations thereof. In some embodiments, CrossMab CH1-CLx The antibody agent comprises a light chain variable domain, said light chain variable domain comprising: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); and (iii) LCDR3 (SEQ ID NO:33). In some embodiments, CrossMab CH1-CLx The antibody agent comprises (a) a heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) a combination thereof; and (b) a light chain variable domain comprising: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); (iii) LCDR3 (SEQ ID NO: 33); or (iv) a combination thereof. In some embodiments, CrossMabCH1-CLx The antibody agent comprises (a) a heavy chain variable domain, the heavy chain variable domain comprising: (i) HCDR1 (SEQ ID NO:18); (ii) HCDR2 (SEQ ID NO:21); and (iii) HCDR3 (SEQ ID NO:24); and (b) a light chain variable domain, the light chain variable domain comprising: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); and (iii) LCDR3 (SEQ ID NO:33).

[0528] In some implementations, the polynucleotides described herein encode CrossMab CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a heavy chain variable domain, and wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); (iii) HCDR3 (SEQ ID NO: 24); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a heavy chain variable domain, and wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO: 18); (ii) HCDR2 (SEQ ID NO: 21); and (iii) HCDR3 (SEQ ID NO: 24). In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a light chain variable domain, and wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); (iii) LCDR3 (SEQ ID NO:33); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a light chain variable domain, and wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO: 27); (ii) LCDR2 (LAS; SEQ ID NO: 30); and (iii) LCDR3 (SEQ ID NO: 33). In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLxThe antibody agent comprises two immunoglobulin chains: a first immunoglobulin chain containing a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO:18); (ii) HCDR2 (SEQ ID NO:21); (iii) HCDR3 (SEQ ID NO:24); or (iv) combinations thereof; and a second immunoglobulin chain containing a light chain variable domain, wherein the light chain variable domain comprises (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); (iii) LCDR3 (SEQ ID NO:33); or (iv) combinations thereof. In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLx The antibody agent comprises two immunoglobulin chains: a first immunoglobulin chain containing a heavy chain variable domain, wherein the heavy chain variable domain comprises: (i) HCDR1 (SEQ ID NO:18); (ii) HCDR2 (SEQ ID NO:21); and (iii) HCDR3 (SEQ ID NO:24); and a second immunoglobulin chain containing a light chain variable domain, wherein the light chain variable domain comprises: (i) LCDR1 (SEQ ID NO:27); (ii) LCDR2 (LAS; SEQ ID NO:30); and (iii) LCDR3 (SEQ ID NO:33).

[0529] In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent comprises a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:36. In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent comprises a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent contains the heavy chain variable domain represented by SEQ ID NO:36. In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLxThe antibody contains the light chain variable domain represented by SEQ ID NO:43.

[0530] In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent comprises a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO:1494. In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent comprises a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody agent contains the heavy chain variable domain represented by SEQ ID NO:1494. In some embodiments, CrossMab is encoded by one or more polynucleotides provided herein. CH1-CLx The antibody contains the light chain variable domain represented by SEQ ID NO:43.

[0531] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., the PGDM1400 antibody agent) comprises a heavy chain variable domain containing one or more mutations relative to the amino acid sequence represented by SEQ ID NO:1494. In some embodiments, a substitution mutation is present at residue 3 and / or 5 relative to SEQ ID NO:1494. In some embodiments, the substitution mutation at residue 3 results in a positively charged amino acid present at residue 3 relative to SEQ ID NO:1494. Positively charged amino acids include Lys(K), Arg(R), and His(H). In some embodiments, the substitution mutation at residue 3 relative to SEQ ID NO:1494 comprises Q3H. In some embodiments, the substitution mutation at residue 5 results in a polar amino acid present at residue 5 relative to SEQ ID NO:1494. Polar amino acids include Ser(S), Thr(T), Tyr(Y), Asn(N), and Gln(Q). In some embodiments, the substitution mutation at residue 5 relative to SEQ ID NO:1494 comprises V5T.

[0532] In some embodiments, the antibody agent encoded by one or more polynucleotides provided herein (e.g., the PGDM1400 antibody agent) includes a heavy chain variable domain containing SEQ ID NO:1494, having substitution mutations Q3H and V5T.

[0533] In some implementations, the polynucleotides described herein encode CrossMab CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a heavy chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 36. In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a light chain variable domain having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence represented by SEQ ID NO:43. In some embodiments, the polynucleotide described herein encodes CrossMab. CH1-CLx The antibody agent contains an immunoglobulin chain, wherein the immunoglobulin chain comprises a heavy chain variable domain as represented by SEQ ID NO:36. In some embodiments, the polynucleotides described herein encode CrossMab. CH1-CLx An immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a light chain variable domain represented by SEQ ID NO:43.

[0534] As described above, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody agent may contain one or more heavy chain constant domains. In some embodiments, the one or more heavy chain constant domains contain a CH3 domain. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody agent contains a CH3 domain, wherein the CH3 domain comprises G1m3, G1m17, or a G1m17,1 allotype. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLxThe antibody agent comprises a CH3 domain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence represented by any one of SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, or 1494. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody agent comprises a CH3 domain having an amino acid sequence represented by any one of SEQ ID NO: 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, or 1495. In some embodiments, the CH3 domain comprises the amino acid sequence SEQ ID NO: 1495, having substitution mutations D16E and L18M.

[0535] CrossMab encoded by one or more polynucleotides as described herein CH1-CLx Antibody agents may comprise one or more heavy chain constant domains, which contain amino acid modifications (e.g., substitutions or deletions) at one or more amino acid positions. For example, CrossMab encoded by one or more polynucleotides as described herein. CH1-CLx Antibody agents may include L / S mutations within the CH3 region (for enhanced FcRn binding) (see Zalevsky J et al., Nat Biotechnol. 2010, which is incorporated herein by reference). Such mutations are designated as M428L and N434S according to EU numbers (i.e., M88L and N94S within the CH3 domain, e.g., SEQ ID NO: 113, 116, or 1495), and are referred to herein as “LS” or “L / S” (see, for example...). Figure 5 (Figure D). In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody contains E294 deletion (for Fc hypersialylation) (see Bas M et al., J Immunol 2019, which is incorporated herein by reference).

[0536] In some implementations, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLxThe antibody agent contains a CH3 domain comprising one or more of the following mutations: Y349C, T366S, L368A, and Y407V (according to EU designation). This combination of mutations is referred to herein as “cah”. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody contains a CH3 domain containing one or more mutations selected from the following: S354C and T366W (according to EU designations). This combination of CH3 mutations is referred to herein as "cak".

[0537] In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: M428L, N434S, or a combination thereof (e.g., an "L / S" mutation). In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence containing any of SEQ ID NO: 117 and 135. In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: Y349C, T366S, L368A, and Y407V (according to EU numbers). In some embodiments, the polynucleotide comprises ribonucleic acid sequences according to SEQ ID NO: 120 and 138. In some embodiments, the polynucleotide encodes a CH3 domain comprising one of the following substitution mutations: S354C and T366W (according to EU numbers). In some embodiments, the polynucleotide comprises ribonucleic acid sequences according to SEQ ID NO: 126 and 144.

[0538] In some embodiments, the polynucleotide encodes an immunoglobulin chain comprising a VH domain operatively linked to one or more constant domains, wherein the one or more constant domains comprise a CH3 domain. In some embodiments, the polynucleotide comprises a ribonucleic acid sequence according to SEQ ID NO: 114. In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence encoding a CH3 domain comprising an allotype of G1m3, G1m17, or G1m17,1. In some embodiments, the polynucleotide comprises a CH3 ribonucleic acid sequence containing either SEQ ID NO: 114 or 132.

[0539] In some implementations, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLxThe antibody agent contains a light chain constant domain. In some embodiments, the light chain constant domain includes a κ light chain constant domain. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody agent comprises a κ light chain constant domain having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO:153. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody agent comprises a κ light chain constant domain having the amino acid sequence represented by SEQ ID NO:153. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx Antibody agents contain a variable λ chain domain.

[0540] In some implementations, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx Antibody agents contain a hinge domain. In some embodiments, CrossMab is encoded by one or more polynucleotides as described herein. CH1-CLx The antibody agent contains a hinge domain comprising the amino acid sequence represented by SEQ ID NO:161 (referred to herein as the “hinge” in Tables 2 and 4).

[0541] In some embodiments, the polynucleotide encodes a hinge domain. In some embodiments, the polynucleotide encodes the hinge ribonucleic acid sequence represented by SEQ ID NO:162. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification comprising the deletion of one or more amino acid residues. In some embodiments, the polynucleotide encodes a hinge domain containing an amino acid modification comprising the deletion of the amino acid residue EPKSC in a conventional Ig hinge domain (represented by SEQ ID NO:161). Such modifications are referred to herein as “hinge_del” or “ΔEPKSC”. In some embodiments, the polynucleotide encodes the hinge ribonucleic acid sequence represented by SEQ ID NO:168. In some embodiments, the polynucleotide encodes a hinge...

Claims

1. A polynucleotide encoding an immunoglobulin chain of an antibody agent, wherein the immunoglobulin chain comprises a single-stranded variable fragment (scFv), and the scFv comprises a heavy chain variable (VH) domain, a linker, and a light chain variable (VL) domain. The VH domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences according to SEQ ID NO:18, 21, and 24, respectively, and wherein the VH domain comprises a first mutation at residue 3 and a second mutation at residue 5 relative to the amino acid sequence according to SEQ ID NO:1494; and The VL domain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1, LCDR2, and LCDR3 comprise amino acid sequences according to SEQ ID NO:27, 30, and 33, respectively.

2. The polynucleotide of claim 1, wherein the first mutation comprises a substitution mutation resulting in a positively charged amino acid at residue 3.

3. The polynucleotide of claim 2, wherein the positively charged amino acid comprises an amino acid selected from Lys(K), Arg(R), or His(H).

4. The polynucleotide of claim 2 or 3, wherein the positively charged amino acid comprises a His(H) residue.

5. The polynucleotide of any one of claims 1-4, wherein the second mutation comprises a substitution mutation resulting in the presence of a polar amino acid at residue 5.

6. The polynucleotide of claim 5, wherein the polar amino acid comprises an amino acid selected from Ser (S), Thr (T), Tyr (Y), Asn (N), or Gln (Q).

7. The polynucleotide of claim 5 or 6, wherein the polar amino acid comprises a Thr(T) residue.

8. The polynucleotide of any one of claims 1-7, wherein the VH domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence according to SEQ ID NO:

36.

9. The polynucleotide of any one of claims 1-8, wherein the VH domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO:

36.

10. The polynucleotide of any one of claims 1-9, wherein the VH domain comprises the amino acid sequence SEQ ID NO:1494, having the substitution mutations Q3H and V5T.

11. The polynucleotide of any one of claims 1-10, wherein the VH domain comprises the amino acid sequence according to SEQ ID NO:

36.

12. The polynucleotide of any one of claims 1-11, wherein the VL domain comprises an amino acid sequence that is at least 85% identical to the amino acid sequence according to SEQ ID NO:

43.

13. The polynucleotide of any one of claims 1-12, wherein the VL domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence according to SEQ ID NO:

43.

14. The polynucleotide of any one of claims 1-13, wherein the VL domain comprises the amino acid sequence according to SEQ ID NO:

43.

15. The polynucleotide of any one of claims 1-14, wherein the scFv comprises, in sequence: (i) The VH structural domain. (ii) the connector, and (iii) The VL structural domain.

16. The polynucleotide of any one of claims 1-14, wherein the scFv comprises, in sequence: (i) The VL structure domain. (ii) the connector, and (iii) The VH structural domain.

17. The polynucleotide of any one of claims 1-16, wherein the linker comprises the amino acid sequence according to SEQ ID NO:

48.

18. The polynucleotide of any one of claims 1-16, wherein the linker comprises the amino acid sequence according to SEQ ID NO:

59.

19. The polynucleotide of any one of claims 1-18, wherein the immunoglobulin chain further comprises a second linker following the scFv domain.

20. The polynucleotide of claim 19, wherein the scFv and the second linker comprise or consist of an amino acid sequence according to SEQ ID NO: 64, 67, 70 or 73.

21. The polynucleotide of any one of claims 1-20, wherein the immunoglobulin chain comprises a hinge domain following the second linker.

22. The polynucleotide of claim 21, wherein the hinge domain comprises or is composed of the amino acid sequence according to SEQ ID NO:

167.

23. The polynucleotide of any one of claims 1-9, wherein the immunoglobulin chain comprises one or more constant domains, and wherein the scFv is operatively linked to the one or more constant domains.

24. The polynucleotide of claim 10, wherein the hinge domain is between the scFv and the one or more constant domains.

25. The polynucleotide of claim 10 or claim 11, wherein one or more constant domains comprise a CH3 domain.

26. The polynucleotide of claim 25, wherein the CH3 domain comprises a G1m17,1 or G1m3 allotype.

27. The polynucleotide of claim 26, wherein the CH3 domain comprises one or more substitution mutations, wherein the one or more substitution mutations comprise or consist of M88L, N94S or a combination thereof, and wherein the substitution mutation position is relative to the amino acid sequence according to SEQ ID NO:1495.

28. The polynucleotide of claim 27, wherein the CH3 domain comprises a substitution mutation at residues 16 and 18 relative to the amino acid sequence according to SEQ ID NO:1495.

29. The polynucleotide of claim 28, wherein the CH3 domain comprises substitution mutations D16E and L18M relative to the amino acid sequence according to SEQ ID NO:1495.

30. The polynucleotide of any one of claims 25-29, wherein the CH3 domain comprises or is composed of the amino acid sequence according to SEQ ID NO:

116.

31. The polynucleotide of claim 1, wherein the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1343.

32. The polynucleotide of claim 1, wherein the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1346.

33. The polynucleotide of claim 1, wherein the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1349.

34. The polynucleotide of claim 1, wherein the immunoglobulin chain comprises or is composed of the sequence according to SEQ ID NO:1352.

35. The polynucleotide of any one of claims 1-34, wherein the polynucleotide comprises a ribonucleic acid sequence encoding a secretion signal.

36. The polynucleotide of claim 35, wherein the secretion signal comprises a ribonucleic acid sequence that is at least 90% identical to that of SEQ ID NO:4 or 8.

37. The polynucleotide of claim 36, wherein the secretion signal comprises a ribonucleic acid sequence comprising SEQ ID NO:4 or 8.

38. The polynucleotide of any one of claims 1-37, wherein the polynucleotide comprises one or more non-coding sequence elements.

39. The polynucleotide of claim 38, wherein one or more non-coding sequence elements enhance RNA stability and / or translation efficiency.

40. The polynucleotide of claim 38 or 39, wherein the one or more non-coding sequence elements comprise a 3' untranslated region (UTR), a 5' UTR, a 5'-cap, a polyadenine (polyA) tail, or any combination thereof.

41. The polynucleotide of claim 40, wherein the polyA tail is or comprises a modified polyA sequence, preferably a discontinuous polyA tail.

42. The polynucleotide of claim 41, wherein the polyA tail comprises or is composed of a sequence that is at least 90% identical to or constitutes the same as SEQ ID NO:

16.

43. The polynucleotide of any one of claims 40-42, wherein the 3'UTR comprises or is composed of a nucleic acid sequence that is at least 90% identical to or constitutes the same as SEQ ID NO:

14.

44. The polynucleotide of any one of claims 40-43, wherein the 5'UTR comprises or is composed of a nucleic acid sequence that is at least 90% identical to or constitutes the same as SEQ ID NO:

10.

45. The polynucleotide of any one of claims 40-44, wherein the 5'-cap is (m2 7,3’ -O)Gppp(m 2’-O )ApG.

46. ​​The polynucleotide of any one of claims 1-45, wherein the polynucleotide comprises one or more modified ribonucleotides.

47. The polynucleotide of claim 46, wherein one or more modified ribonucleotides comprise pseudouridine.

48. The polynucleotide of any one of claims 1-47, wherein the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1000.

49. The polynucleotide of any one of claims 1-47, wherein the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1048.

50. The polynucleotide of any one of claims 1-47, wherein the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1096.

51. The polynucleotide of any one of claims 1-47, wherein the polynucleotide comprises or is composed of the ribonucleic acid sequence according to SEQ ID NO:1144.

52. A polynucleotide comprising or composed of a ribonucleic acid sequence according to any one of SEQ ID NO: 1000, 1048, 1096 or 1044.

53. A composition comprising one or more polynucleotides as described in any one of claims 1-52.

54. The composition of claim 53, wherein the composition further comprises lipid nanoparticles, polymeric complexes (PLX), lipotropic polymeric complexes (LPLX), or liposomes. The one or more polynucleotides therein are wholly or partially encapsulated within the lipid nanoparticles, polymeric complexes (PLX), lipotropic polymeric complexes (LPLX), or liposomes.

55. The composition of claim 53 or claim 54, wherein the composition further comprises lipid nanoparticles. The one or more polynucleotides are encapsulated within the lipid nanoparticles.

56. The composition of claim 54 or claim 55, wherein the lipid nanoparticles are cationic lipid nanoparticles.

57. A pharmaceutical composition comprising the composition as described in any one of claims 53-56 and at least one pharmaceutically acceptable excipient.

58. The pharmaceutical composition of claim 57 for treating and / or preventing HIV, wherein the treatment and / or prevention comprises administering the pharmaceutical composition to a subject.

59. A method comprising administering to a subject the pharmaceutical composition as claimed in claim 57 or claim 58.

60. The method of claim 59 or the pharmaceutical composition for use according to claim 58, wherein administration of the pharmaceutical composition to the subject results in the expression of the antibody agent in the subject.

61. The method of claim 59 or 60, or the pharmaceutical composition for use according to claim 58, wherein the antibody agent is expressed in the subject at the following titers: (a) at least 1 μg / ml in plasma; or (b) At least 1 μg / ml in serum.

62. The method of claim 61, wherein the antibody agent is expressed in the subject at the following titers: (a) at least 10 μg / ml in plasma; or (b) At least 10 μg / ml in serum.

63. The method of claim 62, wherein the antibody agent can be detected in the serum of the subject for a period of at least 5 days, at least 10 days, at least 15 days, at least 20 days, at least 25 days, or at least 30 days.

64. The method of claim 62 or claim 63, wherein the antibody agent is detectable in the serum of the subject for a period of at least 30 days.

65. The method of any one of claims 59-64 or the pharmaceutical composition for use according to claim 58, wherein the antibody is capable of neutralizing one or more HIV strains when tested in a TZM-bl cell pseudovirus neutralization assay at an antibody concentration up to 25 μg / ml.

66. The method of any one of claims 59-65 or the pharmaceutical composition for use according to claim 58, wherein the antibody agent delivered in the form of polynucleotides has higher neutralizing activity against one or more HIV strains compared with an equivalent amount of parental control antibody delivered in the form of polynucleotides, wherein the parental control antibody is an IgG antibody containing the same VH and VL domains as the antibody agent.

67. The method of claim 65 or claim 66, wherein the one or more HIV strains comprise one or more HIV strains selected from the group consisting of: ZM53M.PB12, Du156.12, Q769.d22, 0330.v4.c3, R2184.c04, 89-F1_2_25, CAP204_2_00_F6_6, Ce1176_A3, 6980.v0.c31, PVO.4, CAP45, CNE8, T250-4, 3103.v3.c10, and C1080_c3.

68. The method of any one of claims 59-67 or the pharmaceutical composition for use according to claim 58, wherein the subject is infected with HIV or is at risk of contracting HSV infection.

69. The method of any one of claims 59-68, wherein the method is a method for treating and / or preventing HIV infection.

70. Use of the composition of any one of claims 53-56 or the pharmaceutical composition of claim 57 or claim 58 for the treatment and / or prevention of HIV in a subject.

71. A method of generating an antibody agent, the method comprising administering to cells a composition as described in any one of claims 51-54 or a pharmaceutical composition as described in claim 57 or claim 58, such that the cells express and secrete the antibody agent.

72. The method of claim 71, wherein the cells are in the body of the subject and the antibody agent is produced at a treatment-related plasma concentration or a treatment-related serum concentration.

73. The method of claim 72, wherein the treatment-related plasma concentration or the treatment-related serum concentration is at least 10 μg / ml.

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