Humanized monoclonal antibodies, methods of making and treatment of neisseria gonorrhea

Abstract

Isolated antigen binding polypeptides having a heavy chain variable region and / or light chain variable region that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae as well as antibodies and antigen binding antibody fragments containing the heavy chain variable region and / or the light chain variable region that bind to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae are provided. Pharmaceutical compositions and kits comprising the isolated antigen binding polypeptide and antibodies or antigen binding antibody fragments thereof comprising the antigen binding polypeptide are also provided, along with methods of treating an infection caused by Neisseria gonorrhoeae and methods of making the polypeptides, antibodies and antigen-binding antibody fragments.

Description

HUMANIZED MONOCLONAL ANTIBODIES, METHODS OF MAKING ANDTREATMENT OF NEISSERIA GONORRHEAINCORPORATION OF MATERIAL OF XML SEQUENCE LISTING BY REFERENCE

[0001] The sequence listing submitted herewith as an XML file named “STRXIOOlWOSequenceListing” created on September 1, 2025, which is 60 kilobytes in size, is hereby incorporated by reference in its entirety.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. provisional application no. 63 / 690,898 filed on September 5, 2024, the disclosure of which is hereby incorporated by reference in its entirety' as if fully set forth herein.FIELD OF THE DISCLOSURE

[0003] This disclosure relates to isolated antigen binding polypeptides and antibody or antigen binding antibody fragments having specific binding to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae. This disclosure also relates to methods of making and using the antigen binding polypeptides, antibody fragments and antibodies for treatment of infections caused by Neisseria gonorrhoeae.BACKGROUND OF THE DISCLOSURE

[0004] Each year, there are ~87 million cases of gonorrhea worldwide causing serious sequelae in women, including pelvic inflammatory' disease resulting in tubal factor infertility, ectopic pregnancy, and chronic pelvic pain. In men, gonorrhea causes urethritis, epididymitis and - in men who have sexual relations with other men (MSM) - proctitis. Ceftriaxone is currently the only antibiotic recommended as the first line of treatment of gonorrhea. Rapid spread of azithromycin resistance led to the CDC no longer recommending its use for gonorrhea treatment. However, N. gonorrhoeae isolates that are highly resistant to ceftriaxone have emerged in Asia and Europe and they may soon usher in an era of potentially untreatable gonococcal infection.

[0005] There are no licensed vaccines or biologicals against gonorrhea. A meningococcal vaccine prepared using detergent-extracted outer membrane vesicles (dOMV) from N. meningitidis (MeNZB) was associated with 31% efficacy in gonococcal infection in NewZealand. Recently, in a retrospective observational study conducted in the US, two-dose administration of an FDA-licensed group B meningococcal vaccine (MenB-4C) that contains the same dOMV as MeNZB, led to 40% effectiveness against gonorrhea and a partial MenB- 4C vaccination series was 26% effective. Several other vaccine candidates are being evaluated in early-stage preclinical studies, including gonococcal OMVs, proteins such as MetQ, LptD, BamA, MtrE, and proteins involved in iron or zinc acquisition (e.g. TbpA, TbpB, LbpA, LbpB, ZnuD, TdfH, and TdfJ). There are no licensed biologicals including monoclonal antibodies for gonorrhea.

[0006] Anti-gonococcal lipo-oligosaccharide mAb 2C7 (IgG.G.) was identified by Gulati S. et al., “Immunogenicity' of Neisseria gonorrhoeae lipo-oligosaccharide epitope 2C7, widely expressed in vivo with no immunochemical similarity to human glycosphingolipids'’ [published erratum appears in J. Infect. Dis., 175(4): 1027 (1997)], J. Infect. Dis., 174: 1223- 37 (1996) (hereinafter '‘Gulati Py, the disclosures of which are herein incorporated by reference herein in their entirety. To raise 2C7, mice were hyperimmunized with gonococcal outer membranes and boosted three days prior to fusing splenocytes with Sp2 / 0 myeloma cells. Neisseria gonorrhoeae strains used in this effort were obtained from the collections of the Walter Reed Army Institute of Research and had been extensively characterized. mAb 2C7 was prepared by the method of Kohler G. and Milstein C., “Continuous cultures of fused cells secreting antibody of predefined specificity ’’, Nature, 256:495-7 (1975). w ith the fusions performed by the method of Kennett RH., “Cell fusion”, Methods Enzymol. , 58:345-59 (1979); the disclosures of which are hereby incorporated by reference herein in their entirety. Hybridoma clones secreting anti-lipo-oligosaccharide (LOS) specific antibodies were identified by ELISA and cloned by limiting dilution.

[0007] Further work was undertaken by Yamasaki R. et al. , “Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7; the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes”, J. Biol. Chem., 274:36550-8 (1999), to elucidate the mAb 2C7-defined epitope; the disclosure of which is hereby incorporated by reference herein in its entirety. First, it was found that mAb 2C7 does not bind to the lipid A moiety of the LOS, instead it binds to the oligosaccharide (OS) portion, evidenced by binding to a tyraminated derivative of OS (see Gulati I). Second, 2C7 does not bind LOS lacking OS on the Hep[2], See Banerjee A. et al., “Identification of the gene (IgtG) encoding the lipo- oligosaccharide beta chain synthesizing glucosyl transferase from Neisseria gonorrhoeae'’'. Proc. Natl. Acad. Sci. USA, 95: 10872-7 (1998), the disclosures of which are herebyincorporated by reference herein in their entirety. Using truncated gonococcal LOS structures, the importance of a lactose on Hep[2] for expression of 2C7 epitope was established (see Gulati I). LOS produced by a strain called “WG”, isolated from disseminated gonococcal infection, contains a Gaipi-4GlcNAcpi-3Gal|31-4Glc (lactoneotetraose) and a Gaipi-4Glc (lactose), respectively, on Hep[l] and Hep[2] of the GlcNAc-Hep[2]-Hep[l] trisaccharide core. mAb 2C7 recognizes the inner LOS present in WG LOS (see FIG. 1) and additionally requires N-linked fatty acids in the lipid A for maximum expression of its epitope (see Yamasaki et al.). In short, mAh 2C7 binds to LOS whose OS are elongated from the lactose on Hep[l] of the 15253 OS but not on Hep[2] (see Yamasaki et al.).

[0008] 2C7-negative gonococcal mutants created by deleting IgtG were attenuated in the mouse vaginal colonization model. See Gulati S. et al., “Immunization against a Saccharide Epitope Accelerates Clearance of Experimental Gonococcal Infection”, PLoS Pathog., 9:el003559 (2013) (herein “Gulati IP'), w\A Ram. S. et al., “A novel sialylation site on Neisseria gonorrhoeae lipo-oligosaccharide links heptose II lactose expression with pathogenicity’”. Infect. Immun. 2018:e00285-18 (hereinafter “Rarn Tf, the disclosures of which are hereby incorporated by reference herein in their entirety. As shown in FIG. 2, the IgtG deletion mutant was outcompeted by the wildtype (2C7-positive) isolate (see Gulati II). A second study, using a different gonococcal strain, confirmed significantly decreased colonization by a IgtG deletion mutant (see Ram S. et al.).

[0009] A study conducted by Lam and Gray-Owen aimed to further elucidate potential gonococcal mutations responsible for increased bacterial fitness in vivo. A genome-wide analysis was performed on isolates serially passaged in mice. Standard draft genomes were generated by assembly to a previously annotated and published gonococcal genome. Draft genomes were multi-aligned, mutations were documented, and variants prioritized. A single mutation in IgtG was present in 100% of in vivo passaged isolates and completely absent in parental (clinical isolate N2009) and in vitro passaged isolates. See Lam J., “Genetic adaptation contributing to increased gonococcal fitness during vaginal infection of CEAC AM-humanized mice”. Masters Thesis, Department of Genetics, University of Toronto (2017) (available online): or Lam J. and Gray-Owen S.D., “Genetic adaptation contributing to increased gonococcal fitness during vaginal infection of CEAC AM-humanized mice”, 20th International Pathogenic Neisseria Conference. Manchester, United Kingdom., 2016:24; the disclosures of which are hereby incorporated by reference herein in their entirety. The mutation led to a reading frame shift, resulting in the complete transcription of IgtG (i.e., IgtG‘on’) for in vivo isolates. These data provide further independent verification for the importance of IgtG expression in vivo.

[0010] The 2C7 epitope was expressed by >95% of clinical gonococcal isolates recovered directly from cervical secretions (without sub-passage) from a cohort of women in Boston in the early 1990s (see Gulati I) and by 100% of minimally (<2) sub-passaged isolates from women in Nanjing. China from 2009-2013 (See Ram I). mAb 2C7 exhibits complementdependent bactericidal (killing) activity in vitro against all tested minimally passaged clinical gonococcal isolates (Ram I). The degree of killing of gonococci (or inversely, % survival) by mAb 2C7 is dependent on the relative display of the 2C7 oligosaccharide epitope and consequently the amount of 2C7 binding, (see Fig. 3 from Ram I).

[0011] A study of immunity to urethral reinfection that was carried out in male human volunteers found that the men who resisted N. gonorrhoeae reinfection were significantly more likely to have had a >4x increase in a-LOS IgG following initial infection than those who did not resist reinfection (see Schmidt K.A. et al., “Experimental gonococcal urethritis and reinfection with homologous gonococci in male volunteers”, Sex Transm. Dis.. 28:555-64 (2001) (hereinafter "Schmidt et al.' , the disclosure of which is hereby incorporated by reference herein in its entirety.Establishment of mouse vaginal colonization model to assess mAb 2C7 efficacy.

[0012] The mouse vaginal colonization model of gonorrhea, initially described by Taylor- Robinson et al., "'Neisseria gonorrhoeae colonises the genital tract of oestradiol-treated germ- free female mice”, Microb. Pathog., 9:369-73 (1990) (hereinafter "Taylor-Robinson") and developed further by Jerse (see Jerse A.E. , “Experimental gonococcal genital tract infection and opacity protein expression in estradiol-treated mice”, Infect. Immun., 67:5699-708 (1999) (hereinafter “Jerse ) and Jerse A.E. et al., "Estradiol-Treated Female Mice as Surrogate Hosts for Neisseria gonorrhoeae Genital Tract Infections”, Front. Microbiol. 2: 107 (2011) (hereinafter "Jerse IF); the disclosures of which are hereby incorporated by reference herein in their entirety) is currently the only non-human animal model available for studies of gonorrhea. As such, mAb 2C7 has been evaluated extensively in this model. Briefly, female BALB / c mice 6-8 weeks of age in the diestrus phase of the estrous cycle were started on treatment (that day) with 0.5 mg Premarin in 200 pl water given subcutaneously on each of 3 d; -2, 0, and +2 d (before, the day of, and after inoculation) to prolong the estrus phase of the cycle and promote susceptibility to N. gonorrhoeae infection. Premarin is a mixture of sodium estrone sulfate and sodium equilin sulfate and as concomitant components, sodiumsulfate conjugates of 17a-dihydroequilin, 17a-estradiol, and 17p-dihydroequilin. Mice were administered vancomycin (0.6 mg) and streptomycin sulfate (0.3 mg) intraperitoneally on each of 3 d; -2, -1, and 0 d (before and the day of inoculation) to reduce competitive microflora. The inoculum size was specified for each experiment. Daily bacterial burdens were measured by enumerating CFU obtained by first ringing vaginal swabs in 100 pl of nonnal saline and then plating serial 10-fold dilutions onto chocolate agar plates containing IsoVital ex equivalent and VCNTS (vancomycin, colistin, nystatin, and trimethoprim sulfate) supplement plus 100 mg of streptomycin sulfate per ml of media. Fig. 4 shows a schematic that outlines the procedures used in this model.Efficacy of intraperitoneally administered murine mAb 2C7 against N. gonorrhoeae.

[0013] The passive administration of murine mAb 2C7 was examined for its effect on shortening the time of N. gonorrhoeae strain FA1090 infection in mice. See Gulati II. Mice were administered mAb 2C7 or control IgG.A mAb intraperitoneally daily for 3 days prior to and including the day of challenge. In Group 1 (Figs. 5A-5C, graphs on left), using a vaginal inoculum of 5x 105CFU, 16 of 20 (80%) mice administered mAb 2C7 and 16 of 20 mice administered control IgGA mAb became infected. In Group 2 (Fig. 5A-5C, graphs on right), using a 3.6*105CFU inoculum; 18 of 20 (90%) mice administered mAb 2C7 and 18 of 20 mice given control IgG.A mAb became infected. Kaplan Meier analysis showed that mAb 2C7-treated animals cleared gonococci faster than IgG.A mAb-treated control animals in both groups (Group 1 : median time to clearance; 4 days in mAb 2C7 administered vs. 6 days in the IgG.A mAb administered group, P=0.03 [FIG 5A, left panel]; Group 2: median time to clearance; 6 days in mAb 2C7 administered vs. 9 days in the IgG.A mAb administered group, P=0.008 [Fig. 5A, right panel]). A diminished longitudinal trend in mean logio CFU colonization over time was measured in mAb 2C7 administered mice (P=0.008 and P=0.0005 in Groups 1 and 2, respectively [Fig. 5B]). Cumulative CFU expressed as area under the curve (AUC) was also lower in mAb 2C7 administered mice (P=0.014 and P=0.0114 in Groups 1 and 2, respectively [Fig. 5C]). All gonococcal colonies isolated from the 2 mice that remained infected at 7 days in Group 2 after inoculation (75 and 40 colonies per mouse) were detected by mAb 2C7 in colony blots further emphasizing that the 2C7 epitope while important for prolonged survival of gonococci in the mouse model (Gulati II) also does not “phase-vary off’ with selective pressure applied by mAb 2C7.SUMMARY OF THE DISCLOSURE

[0014] In one aspect, the present invention provides an isolated antigen binding polypeptide that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae. The polypeptide comprises a heavy chain variable region including a human framework region and three non-human complementarity7determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the human framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS(SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3); wherein Xi is N or Q or S or A; X2is N or Q or S or A; X3 is N or Q or S or A, X4is W or H or Y or F. The lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae is 2C7.

[0015] In another aspect, the present invention provides an isolated antigen binding polypeptide that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a light chain variable region including a human framework region and three non-human complementarity7determining regions (CDRs) having sequences LI, L2. and L3 incorporated into the human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4); the L2 sequence is GIX6X7RAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXIO (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xe is N or Q or S or A; X7 is N or Q or S or A, Xs is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y. The lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae is 2C7.

[0016] In another aspect, the present invention includes a product formed by a combination of any of the above-described isolated antigen binding polypeptides. The isolated antigen binding polypeptide that binds specifically to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprises a heavy chain variable region including a first human framework region and three non-human complementarity determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the first human acceptor framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS (SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3);wherein Xi is N or Q or S or A; X2 is N or Q or S or A; X3 is N or Q or S or A, X4 is W or H or Y or F. in combination with a light chain variable region including a second human framework region and three non-human CDRs having sequences LI, L2, and L3 incorporated into the second human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4); the L2 sequence is GIXgX RAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXio (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xg is N or Q or S or A; X7 is N or Q or S or A, X8is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y. The first human framework region and the second human framework region are different. The lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae is 2C7.

[0017] In each of the previous embodiments of paragraphs

[0014] and

[0016] , the Hl sequence may be GYTFTDYNME (SEQ ID NO: 7), the H2 sequence may be VINPNNRFTS (SEQ ID NO: 8) and the H3 sequence may be SRWYQYDY (SEQ ID NO: 9).

[0018] In each of the previous embodiments of paragraphs

[0015] to

[0016] , the LI sequence may be RSSTGAVTTSNYAN (SEQ ID NO: 10), the L2 sequence may be GINNRAP (SEQ ID NO: 11) and the L3 sequence may be ALWYSNHWV (SEQ ID NO: 12).

[0019] In each of the previous embodiments of paragraphs

[0014] and

[0016] to

[0018] , the human framework region of the heavy chain or the first human acceptor framework region outside of the Hl, H2, and H3 sequences in the heavy chain variable region, may be derived from an amino acid sequence of SEQ ID NO: 13.

[0020] In the previous embodiment of paragraph

[0019] , the heavy chain variable region may have at least one amino acid substitution selected from the group consisting of Q1E, V5Q, A9P, V11L, K12V, A16S, V20I, G24A, R38K, A40S, P41H, Q43K, G44S, M48I, A61N, K63N, Q65R, R67K, V68A, M70L, R72V, T74K, T76S, V79A, E82D, S84R, R87T, T91S, Y95F, R98G, and V113L.

[0021] In the previous embodiment of paragraph

[0020] , the heavy chain variable region may have at least one amino acid substitution selected from the group consisting of Q1E, V5Q, K12V, V20I, G24A, M48I, V68A, M70L, R72V, T74K, V79A, R98G, and V113L.

[0022] In each of the previous embodiments of paragraphs

[0014] and

[0016] to

[0021] , the heavy chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22.

[0023] In each of the previous embodiments of paragraphs

[0015] to

[0016] , and

[0018] , the human framework region of the light chain or the second human acceptor framework outside of the LI, L2, and L3 sequences in the light chain variable region, may be derived from an amino acid sequence of SEQ ID NO: 25.

[0024] In the previous embodiment of paragraph

[0023] , the light chain variable region may have at least one amino acid substitution selected from the group consisting of S1Q. S2V, E3V, L4V, D7E, P8S. VIOL, S 11T. V12T, A13S. P14P, Q16E. R19,T, I20L. Y38V, Q40E, G43D, Q44H, A45L, P46F, V47T, L48G, V49L, Y51 G, I60V, D62A, S68L, S69I, N71D, T72A, S74A, A82T, D87I, Y89F, and C90F.

[0025] In the previous embodiment of paragraph

[0024] , the light chain variable region may have at least one amino acid substitution selected from the group consisting of S2V. L4V, VIOL, V12T, P14P, I20L, Y38V, Q40E, Q44H, P46F, L48G, V49L, Y51G, I60V, S68L, and N71D.

[0026] In each of the previous embodiments of paragraphs

[0015] to

[0016] and

[0023] to

[0025] , the light chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-33.

[0027] In each of the previous embodiments of paragraphs

[0014] to

[0026] , the heavy chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22, and the light chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NO: 26-33.

[0028] In the previous embodiment of paragraph

[0014] , the isolated antigen binding polypeptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-43

[0029] In the previous embodiment of paragraph

[0015] , the isolated antigen binding polypeptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-53.

[0030] In another aspect, the present invention provides a humanized antibody or antigen binding antibody fragment comprising the isolated antigen binding polypeptide of any one of paragraphs

[0014] to

[0029] ,

[0031] In one embodiment of paragraph

[0030] , the humanized antibody or antigen binding antibody fragment has a higher binding affinity to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae in comparison to a binding affinity of a chimeric antibody or antigen binding antibody fragment to the LOS epitope of Neisseria gonorrhoeae.

[0032] In each of the previous embodiments of paragraphs

[0030] -

[0031] , the humanized antibody or antigen binding antibody fragment has a binding affinity to the LOS epitope that is at least 2-4 times greater than the binding affinity of the chimeric antibody or antigen binding antibody fragment to the LOS epitope of Neisseria gonorrhoeae.

[0033] In each of the previous embodiments of paragraphs

[0030] -

[0032] , the humanized antibody or antigen binding antibody fragment has at least one additional mutation in the Fc region and has an altered binding affinity for FcRn.

[0034] In the previous embodiment of paragraph

[0033] , the at least one mutation in the Fc region is at one or more positions selected from the group consisting of 311, 428, and 434.

[0035] In each of the previous embodiments of paragraphs

[0033] -

[0034] , the humanized antibody or antigen binding antibody fragment has at least one additional mutation in the Fc region is selected from the group consisting of Q311 R, M428E, and N434W.

[0036] In each of the previous embodiments of paragraphs

[0033] -

[0035] , the humanized antibody or antigen binding antibody fragment has an Fc region comprising the amino acid substitutions Q311R. M428E, and N434W.

[0037] In the previous embodiment of paragraph

[0036] , the Fc region comprises the amino acid sequence of SEQ ID NO: 55.

[0038] In yet another aspect, the present invention provides a pharmaceutical composition comprising the isolated antigen binding polypeptide of any one of paragraphs

[0014] -

[0029] , or the humanized antibody or antigen binding antibody fragment of any one of [paragraphs

[0030] -

[0037] , and a pharmaceutically acceptable carrier.

[0039] In one embodiment, a vaccine is provided comprising the composition of paragraph

[0037] ,

[0040] In another aspect, the present invention provides a method of treating an infection comprising a step of administering the isolated antigen binding polypeptide of any one of paragraphs

[0014] to

[0029] , or the humanized antibody or antigen binding antibody fragment of any one of paragraphs

[0030] to [

[0037] , the pharmaceutical composition of paragraph

[0038] , or the vaccine of

[0039] , to a patient with the infection. The infection may be an infection caused by Neisseria gonorrhoeae.

[0041] In another aspect, the present invention provides use of the isolated antigen binding polypeptide in each of the previous embodiments in paragraphs

[0014] -

[0029] , use of the humanized antibody or antigen binding antigen binding antibody fragment in each of the previous embodiments of paragraphs

[0030] -

[0037] , use of the pharmaceutical compositionof paragraph

[0038] , or use of the vaccine of paragraph

[0039] , for the treatment of an infection. The infection may be an infection caused by Neisseria gonorrhoeae.

[0042] In yet another aspect, the present invention provides a kit including any of the isolated antigen binding polypeptides, the humanized antibody or antigen binding antibody fragment thereof, the pharmaceutical composition, or the vaccine described above, for treatment of an infection. The infection may be an infection caused by Neisseria gonorrhoeae.

[0043] In another aspect, the present invention provides a method of making any of the isolated antigen binding polypeptides or antibody and antigen binding antibody fragments thereof described above, comprising introducing one or more non-human complementarity determining regions (CDRs) into human framework region(s).BRIEF DESCRIPTION OF DRAWINGS

[0044] FIG. 1 shows a generalized structure of N. gonorrhoeae LOS. Linkages show n with an asterisk (*) indicate glycans added by phase variable LOS glycosyltransferases. The epitope recognized by 2C7 is shown in the box indicated by an arrow. mAb 2C7 recognizes the structure shown in the box indicated by the arrow (inner anti-hpo-oligosaccharide (LOS) present in WG LOS). Glycan extensions beyond lactose (Gal-Glc) from HepI permit mAb 2C27 binding. The phase-variable IgtG gene adds Glc to HepII.

[0045] FIGS. 2A-2C show selective survival for FA I 090w t (1.5xl05CFU) andFA I ()90 / g / G ~ (L6xlO5CFU) mixed in equal proportions and inoculated into the mice from Gulati II.

[0046] FIG. 2A shows a Kaplan Meier analysis of time to clearance showing differences in clearance of FA1090wt (solid line) and FA l ()90 / / G (dotted line) mixed together.

[0047] FIG. 2B shows colonization (Logio CFU) at daily intervals of FA1090wt and FA 1090 / / G ~ , limit of detection <5 CFUs.

[0048] FIG. 2C shows bacterial burdens consolidated over time (Area Under the Curve analysis) of FA 1090 t and FA1090 / gtG".

[0049] FIG. 3 shows complement-dependent bactericidal activity of mAb 2C7 against 62 randomly selected isolates collected from men with urethritis in a Nanjing (China) study of gonococcal transmission from men to women as a function of mAb 2C7 binding (expressed as OD450). Bacteria w ere grown in medium containing CMP-Neu5Ac (2 Survival of bacteria at 30 min. following incubation with mAb 2C7 (5 pg / ml) plus normal human serum (NHS;16.7%) as a source of complement (C) is shown as a function of mAb 2C7 binding (X-axis). See Ram I.

[0050] FIG. 4 shows a schematic outlining the procedures for the mouse intravaginal colonization model described by Jerse (above).

[0051] FIGS. 5A-5C show survival of FA1090 wild-ty pe (wt) inoculated into mice passively immunized with mAb 2C7 as demonstrated in Gulati IL mAb 2C7 (dashed lines in FIGS.5A and FIG 5B) vs. control IgG3 mAb (solid lines) treated animals followed by challenge with FAlOOOyvt (~5 x 10’ CFU).. Two separate experiments were performed; Group 1 and Group followed by challenge with FA1090wt (~5 x I05CFU)2 (n=number of infected mice in each group), as indicated.

[0052] FIG. 5A shows a Kaplan Meier analysis of time to clearance of FA1090wt showing differences in clearance of mAb 2C7 vs. control IgG3Z mAb treated animals.

[0053] FIG. 5B shows colonization of FA1090wt (Logio CFU) measured daily in mAb 2C7 vs. control IgG3Z mAb treated animals.

[0054] FIG. 5C shows bacterial burdens consolidated over time (Area Under the Curve [logio CFU] analysis) of mAb 2C7 vs. control lgG3Z mAb treated animals.

[0055] FIG. 6 shows a Western blot of humanized IgG molecules (H2C7-1 through H2C7-8) in the tissue culture supernatants.

[0056] FIGS. 7A-7I show binding assays conducted for H2C7 mAbs 1-8 and chim 2C7 groups that were either undiluted (indicated by the group with the asterisk (*)) or at a 1 / 10 dilution in PBS / Tween (indicated by the group with no asterisk) to bacterial lysates by ELISA.

[0057] FIG. 8 shows binding of H2C7 mAbs 1-8 and chim 2C7 groups to CP2-biotin, a peptide mimic of the 2C7 epitope, that were either undiluted (indicated by the group with the asterisk (*)) or at a 1 / 10 dilution in PBS / Tween (indicated by the group with no asterisk).

[0058] FIGS. 9A-9F show binding of for H2C7 mAbs 1-8 to bacterial lysates by ELISA. FIGS. 9A-9B show' binding to MS 4Hex / G+, while FIGS. 9C-9D show binding to MS 2Hex / G+ on plate 2. Binding of chimeric mAb 2C7 (concentrations indicated on the X-axis) were also included as controls - binding to MS 11 4-Hex / G+ and MS 11 2-Hex / G+ are shown in panels 9E and 9F, respectively.

[0059] FIG. 10 shows a binding assay ofH2C7 mAbs 1-8 versus MS114Hex / G+.

[0060] FIG. 11 shows an assay of H2C7 mAbs 1-8 and Chim. 2C7 versus MS 11 4Hex / G+.

[0061] FIG. 12 shows the serum bactericidal activity of purified humanized mAbs H2C7-6 to H2C7-8 and Chim. 2C7 versus N. gonorrhoeae 15253.

[0062] FIGS. 13A-13B show bactericidal activity of humanized mAh H2C7-8 and three additional derivatives (H2C7-9, H2C7-10 and H2C7-11) against A. gonorrhoeae strains FA1090 (Fig. 14A) and 15253 (Fig. 14B). Complement dependent bactericidal assays were performed using IgG and IgM depleted normal human serum (Pel-Freez) as the complement source. The X-axis is the concentration of mAh used in the assay, and the Y-axis is the % survival of bacteria at 30 min relative to CFUs at 0 min in each of FIGS. 13A-13B.

[0063] FIG. 14 shows 2C7VH sequences of humanized versions of the 2C7 antibody.

[0064] FIG. 15 shows 2C7VL sequences of humanized versions of the 2C7 antibody.

[0065] FIG. 16 shows a sequence alignment of the humanized 2C7 VH regions.

[0066] FIG. 17 shows a sequence alignment of the humanized 2C7 VL regions.

[0067] FIG. 18 shows a sequence alignment of humanized sequences of 2C7 antibodies (full- length) human IgGl / kappa.

[0068] FIG. 19A shows bactericidal activity of STX-426 against N. gonorrhoeae strain FA 1090.

[0069] FIG. 19A shows bactericidal activity of STX-426 against N. gonorrhoeae strain 15253.

[0070] FIG. 20 shows a sequence alignment of each of IgGl WT and REW-Fc (Q311 R / N434W / M428E).REFERENCES

[0071] Gulati S, McQuillen DP, Mandrell RE, Jani DB, Rice PA. Immunogenicity of Neisseria gonorrhoeae lipo-oligosaccharide epitope 2C7, widely expressed in vivo with no immunochemical similarity to human glycosphingolipids [published erratum appears in J Infect Dis 1997 Apr; 175 (4): 1027], J Infect Dis 1996; 174: 1223-37.

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[0074] Yamasaki R, Koshino H, Kurono S, NishinakaY, McQuillen DP, Kume A, Gulati S, Rice PA. Structural and immunochemical characterization of a Neisseria gonorrhoeae epitope defined by a monoclonal antibody 2C7; the antibody recognizes a conserved epitope on specific lipo-oligosaccharides in spite of the presence of human carbohydrate epitopes. J Biol Chem 1999; 274:36550-8.

[0075] Banerjee A. Wang R, Uljon SN, Rice PA, Gotschlich EC, Stein DC. Identification of the gene (IgtG) encoding the lipo-oligosaccharide beta chain synthesizing glucosyl transferase from Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 1998; 95: 10872-7.

[0076] Gulati S, Zheng B, Reed GW, Su X, Cox AD, St Michael F, Stupak J, Lewis LA, Ram S, Rice PA. Immunization against a Saccharide Epitope Accelerates Clearance of Experimental Gonococcal Infection. PLoS Pathog 2013; 9:el003559.

[0077] Ram S. Gulati S, Lewis LA, Chakraborti S, Zheng B, DeOliveira RB. Reed GW, Cox AD, Li J, St Michael F, et al. A novel sialylation site on Neisseria gonorrhoeae lipo- oligosaccharide links heptose II lactose expression with pathogenicity. Infect Immun 2018:e00285-18.

[0078] Lam J, Gray-Owen SD. Genetic adaptation contributing to increased gonococcal fitness during vaginal infection of CEACAM-humanized mice. 20th International Pathogenic Neisseria Conference. Manchester, United Kingdom., 2016:24.

[0079] Gulati S, Beurskens FJ, de Kreuk BJ, Roza M, Zheng B, DeOliveira RB, Shaughnessy J. Nowak NA, Taylor RP, Botto M, et al. Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody. PLoS Biol 2019; 17:e3000323.

[0080] Schmidt KA, Schneider H, Lindstrom JA, Boslego JW, Warren RA, Van de Verg L, Deal CD, McClain JB, Griffiss JM. Experimental gonococcal urethritis and reinfection with homologous gonococci in male volunteers. Sex Transm Dis 2001; 28:555-64.

[0081] Taylor-Robinson D, Furr PM, Hetherington CM. Neisseria gonorrhoeae colonises the genital tract of oestradiol-treated germ-free female mice. Microb Pathog 1990; 9:369-73.

[0082] Jerse AE. Experimental gonococcal genital tract infection and opacity' protein expression in estradiol-treated mice. Infect Immun 1999; 67:5699-708.

[0083] Jerse AE, Wu H, Packiam M, Vonck RA, Begum AA, Garvin LE. Estradiol-Treated Female Mice as Surrogate Hosts for Neisseria gonorrhoeae Genital Tract Infections. Front Microbiol 2011; 2: 107.

[0084] Ram S, Cullinane M, BiomA, Gulati S, McQuillen D, Monks B, O'Connell C, Boden R, Elkins C, Pangbum M, et al. Binding of C4b-binding Protein to Porin: A molecular mechanism of serum resistance of Neisseria gonorrhoeae. J Exp Med 2001; 193:281-96.

[0085] Ram S, McQuillen DP, Gulati S, Elkins C, Pangbum MK, Rice PA. Binding of complement factor H to loop 5 of porin protein 1 A: a molecular mechanism of serum resistance of nonsialylated Neisseria gonorrhoeae. J Exp Med 1998; 188:671-80.

[0086] Ram S, Shanna AK, Simpson SD, Gulati S, McQuillen DP, Pangbum MK, Rice PA. A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae . J Exp Med 1998; 187:743-52.

[0087] Ngampasutadol J, Ram S, Blom AM, Jarva H, Jerse AE, Lien E, Goguen J, Gulati S, Rice PA. Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection. Proc Natl Acad Sci U S A 2005; 102:17142-7.

[0088] Ngampasutadol J, Ram S, Gulati S, Agarwal S, Li C, Visintin A, Monks B, Madico G, Rice PA. Human Factor H Interacts Selectively with Neisseria gonorrhoeae and Results in Species-Specific Complement Evasion. J Immunol 2008; 180:3426-35.

[0089] Ermert D, Shaughnessy J, Joeris T, Kaplan J, Pang CJ, Kurt-Jones EA, Rice PA, Ram S, Blom AM. Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors. PLoS pathogens 2015; 11 :e 1005043.

[0090] Landig CS, Hazel A, Kellman BP, Fong JJ, Schwarz F, Agarwal S, Varki N, Massari P, Lewis NE, Ram S, et al. Evolution of the exclusively human pathogen Neisseria gonorrhoeae: Human-specific engagement of immunoregulalory Siglecs. Evol Appl 2019; 12:337-49.

[0091] Lewis LA, Gulati S, Zelek WM, Morgan BP, Song WC, Zheng B, Nowak N, DeOliveira RB,B, DeSouza Silva L, et al. Efficacy of an Experimental Gonococcal Lipo- oligosaccharide Mimitope Vaccine Requires Terminal Complement. J Infect Dis 2022; 225:1861-4.

[0092] Agarwal S. Vasudhev S, DeOliveira RB. Ram S. Inhibition of the classical pathway of complement by meningococcal capsular polysaccharides. J Immunol 2014; 193: 1855-63.

[0093] Abara WE, Bernstein KT, Le is FMT, Schillinger JA, Feemster K, Pathela P, Hariri S, Islam A, Eberhart M, Cheng I, et al. Effectiveness of a serogroup B outer membrane vesicle meningococcal vaccine against gonorrhoea: a retrospective observational study. Lancet Infect Dis 2022; 22: 1021-9.

[0094] Creighton S, Tenant-Flowers M, Taylor CB, Miller R, Low N. Co-infection with gonorrhoea and chlamydia: how much is there and what does it mean? Int J STD AIDS 2003; 14: 109-13.

[0095] Forward KR. Risk of coinfection with Chlamydia trachomatis and Neisseria gonorrhoeae in Nova Scotia. Can J Infect Dis Med Microbiol 2010; 21:e84-6.

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[0097] Petousis-Harris H, Paynter J, Morgan J, Saxton P, McArdle B, Goodyear-Smith F, Black S. Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study. Lancet 2017; 390: 1603-10.

[0098] Vonck RA, Darville T, O'Connell CM, Jerse AE. Chlamydial infection increases gonococcal colonization in a novel murine coinfection model. Infect Immun 2011; 79: 1566- 77.

[0099] de Jong RN, Beurskens FJ. Verploegen S, Strumane K, van Kampen MD. Voorhorst M, Horstman W, Engelberts PJ, Oostindie SC, Wang G, et al. A Novel Platform for the Potentiation of Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG Hexamers at the Cell Surface. PLoS Biol 2016; 14:el002344.

[0100] Stapleton NM, Andersen JT, Stemerding AM, Bjamarson SP, Verheul RC, Gerritsen J, Zhao Y, Kleijer M, Sandlie I, de Haas M, et al. Competition for FcRn-mediated transport gives rise to short half-life of human IgG3 and offers therapeutic potential. Nat Commun 2011; 2:599.

[0101] Shaughnessy J, Tran Y, Zheng B, DeOliveira RB, Gulati S, Song WC, Maclean JM, Wycoff KL, Ram S. Development of Complement Factor H-Based Immunotherapeutic Molecules in Tobacco Plants Against Multidrug-Resistant Neisseria gonorrhoeae. Front Immunol 2020; 11:583305.

[0102] Chu TH, Patz EF, Jr., Ackerman ME. Coming together at the hinges: Therapeutic prospects of !gG3. MAbs 2021; 13: 1882028.

[0103] Werner A, Nimmerjahn F. HINGEneering IgG for enhanced immune activation. Sci Immunol 2022; 7:eabq4797.

[0104] Roopenian DC, Christianson GJ, Proetzel G, Sproule TJ. Human FcRn Transgenic Mice for Pharmacokinetic Evaluation of Therapeutic Antibodies. Methods Mol Biol 2016; 1438: 103-14.

[0105] Hosseini I, Gajjala A, Bumbaca Yadav D, Sukumaran S, Ramanujan S, Paxson R, Gadkar K. gPKPDSim: a SimBiology((R))-based GUI application for PKPD modeling in drug development. J Pharmacokinet Pharmacodyn 2018; 45:259-75.

[0106] Grevys A, Nilsen J, Sand KMK, Daba MB, Oynebraten I, Bern M, McAdam MB, Foss S, Schlothauer T, Michaelsen TE, et al. A human endothelial cell-based recycling assay for screening FcRn targeted molecules. Nat Commun 2018; 9:621.

[0107] See Lam J., “Genetic adaptation contributing to increased gonococcal fitness during vaginal infection of CEACAM-humanized mice". Masters Thesis, Department of Genetics, University of Toronto (2017) (available online), o Lam J. and Gray-Owen S.D., “Geneticadaptation contributing to increased gonococcal fitness during vaginal infection of CEACAM-humanized mice’', 20th International Pathogenic Neisseria Conference. Manchester, United Kingdom., 2016:24.

[0108] Foss, S. et al., “Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria”, Nat. Commun., 15:2007 (2024) (https: / / doi.org / 10.1038 / s41467-024-46321-9).DEFINITIONS

[0109] In order to facilitate understanding of the examples provided herein, certain frequently occurring terms are defined herein.

[0110] In connection with a measured quantity, the term "about" as used herein refers to the normal variation in that measured quantity that would be expected by a skilled person making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Unless otherwise indicated herein or recognized as the normal variation for the particular measured quantity, "about" refers to a variation of + / - 10% of the value provided.[OHl] The term '‘affinity” as used herein refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described herein.

[0112] The term "amino acid" as used herein refers to any organic compound that contains an amino group (-NH2) and a carboxyl group (— COOH); preferably either as free groups or alternatively after condensation as part of peptide bonds. The "twenty naturally encoded polypeptide-forming alpha-amino acids" are understood in the art and refer to: alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or C). gluatamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), try ptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0113] The term “antibody” as used herein refers to intact immunoglobulin molecules. Antibodies can be used to isolate preparative quantities of the antigen by immunoaffinity chromatography. Various other uses of such antibodies are to diagnose and / or stage disease (e.g., neoplasia) and for therapeutic application to treat disease, such as for example: neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections, and the like. Chimeric, human-like, humanized or fully human antibodies are particularly useful for administration to human patients. An antibody comprises an antigen-binding variable region (VH or VL) as well as a light chain constant domain (CL) and heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different “classes”. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0114] The term “humanized antibody” refers to intact immunoglobulin molecules, that are capable of binding to an epitope of an antigen and having an antigen binding site derived from an immunoglobulin from a non-human species, and remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The antigen binding site typically comprises complementarity determining regions (CDRs) which determine the binding specificity of the antibody molecule, and which are carried on appropriate framework regions in the variable domains. There are 3 CDRs (CDR1. CDR2 and CDR3) in each of the heavy and light chain variable domains.

[0115] The term “humanized antibody fragment” refers to fragments of immunoglobulin molecules, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen and having an antigen binding site derived from an immunoglobulin from a non-human species, and remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin.

[0116] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of a whole antibody molecule with the enzy me papain, to yield a fragment consisting of an intact light chain and a portion of a heavy’ chain.

[0117] An Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.

[0118] An (Fab')2 fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fab')2 fragment is a dimer of tw o Fab' fragments, held together by two disulfide bonds.

[0119] An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as tw o chains.

[0120] An Fc region is defined as a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term “Fc region” includes native Fc regions and variant Fc regions. For instance, with respect to an IgGl, the antibody comprises an antigen-binding variable region (VH or VL) as w ell as a light chain constant domain (CL) and heavy chain constant domains (CHI, CH2 and CH3). The heavy chain constant domains CH2 and CH3 form the Fc region of an IgGl.

[0121] The term “antibody fragment” as used herein refers to a molecule other than an intact immunoglobulin molecule that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to fragments of immunoglobulin molecules such as Fv, Fab. Fab'. Fab'-SH. F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments that are capable of binding to an epitope of an antigen. These antibody fragments, which retain some ability to selectively bind to an antigen (e.g., a polypeptide antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e.g., Harlow and Lane, supra), and are described further, as follows.

[0122] The phrases “% identity” or “percent identity” or “percent sequence identity” or “PID” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of nucleic acid residues or amino acid residues, respectively, that are the same, when compared and aligned for maximum similarity, as determined using a sequence comparison algorithm or by visual inspection. Percent identity may be determined by using standard techniques known in the art. The percentage amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using aprogram such as BLAST, MUSCLE, or CLUSTAL. The BLAST algorithm is described, for example, mAltschul et al.. J. Mol. Biol., 215:403-410 (1990) and Karlin etal., Proc Natl Acad Sci USA, 90:5873-5787(1993). A percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference’' sequence including any gaps created by the program for optimal / maximum alignment. BLAST algorithms refer to the “reference’' sequence as the “query” sequence. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1992)) alignments (B) of 50, expectation (E) of 10, M'5, N'-4. and a comparison of both strands.

[0123] Percent sequence identify” or “% identity” or “% sequence identity or “% amino acid sequence identify” of a subject amino acid sequence to a reference (i.e., query) amino acid sequence means that the subject amino acid sequence is identical (i.e., on an amino acid-by- amino acid basis) by a specified percentage to the query amino acid sequence over a comparison length when the sequences are optimally aligned. Thus, 80% amino acid sequence identify or 80% identity with respect to two amino acid sequences means that 80% of the amino acid residues in two optimally aligned amino acid sequences are identical.

[0124] A polypeptide of interest may be said to be “substantially identical” to a parent polypeptide if the polypeptide of interest comprises an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. at least about 99%, or at least about 99.5% sequence identity to the amino acid sequence of the parent polypeptide. The percentage identity between two such polypeptidescan be determined manually by inspection of the two optimally aligned polypeptide sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative amino acid substitution or one or more conservative amino acid substitutions.

[0125] The term “effective amount” of one or more polypeptide variants described herein refers to the amount of polypeptide, antibody or antibody fragment that achieves desired level of activity in a specific composition, such as but not limited to a bactericidal composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular application, the specific composition of the bactericidal composition, and whether a liquid or dry (e.g., granular, tablet, bar, pods) or single-unit dose composition is required.

[0126] The term "binding" as used herein refers to interaction of the variable region or an Fv of an antibody with an antigen with the interaction depending upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure rather than to proteins generally. As used herein, the tenn "specifically binding" or "binding specifically" means that an antibody variable region or Fv binds to or associates with more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen than with other proteins. For example, an antibody variable region or Fv specifically binds to its antigen with greater affinity, avidity, more readily, and / or with greater duration than it binds to other antigens. For another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with materially greater affinity’ than it does to related proteins or other cell surface proteins or to antigens commonly recognized by polyreactive natural antibodies (i.e., by naturally occurring antibodies known to bind a variety of antigens naturally found in humans). However, "specifically binding" does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term "selective binding".

[0127] The term “framework” or “FR” as used herein refers to variable domain residues other than complementarity determining regions (CDRs or Hl -3 in the heavy chain and LI -3 in the light chain) residues. The FR of a variable domain generally consists of four FRdomains: FR1, FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0128] The term '‘isolated” polypeptide or antibody as used herein is one which has been separated from a component of its natural environment. In some embodiments, an isolated polypeptide or antibody is purified to greater than 95% or 99% purity7as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g.. ion exchange or reverse phase High Performance Liquid Chromatography (HPLC)). For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0129] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally7occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance wi th the present invention may be made by a variety7of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phagedisplay methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0130] The term “therapeutically effective amount” of the polypeptide, antibody, antibody fragment or compositions of the invention refers to a sufficient amount to treat an infection, at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety7of factors including the infection being treated and the severity7of the infection; activity of the specific antibody employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific antibodyemployed; the duration of the treatment; drugs used in combination or coincidental with the specific antibody employed; and like factors well known in the medical arts.

[0131] The term '‘treatment;’ "treat." or '‘treating” as used herein refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology7. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of infection, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the infection, , decreasing the rate of infection progression, amelioration or palliation of the infection. In some embodiments, antibodies of the invention are used to delay development of an infection or to slow the progression of the infection.

[0132] The term “variable region” or “variable domain” as used herein refers to the domain of a heavy or light chain that is involved in binding to the antigen. The variable domains of the heavy7chain and light chain (VH and VL, respectively) generally7have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, polypeptides, antibodies or antibody fragments that bind a particular antigen may be isolated using a VH or VL domain that binds the antigen to screen a library7of complementary7VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol., vol. 150. pp. 880-887. 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.DETAILED DESCRIPTION

[0133] For illustrative purposes, the principles of the present invention are described by referencing various exemplary embodiments. Although certain embodiments of the invention are specifically7described herein, one of ordinary skill in the art will readily recognize that the same principles are equally applicable to, and can be employed in, other systems and methods. Before explaining the disclosed embodiments of the present invention in detail, it is to be understood that the invention is not limited in its application to the details of any particular embodiment shown. Additionally, the terminology used herein is for the purpose of description and not for limitation. Furthermore, although certain methods are described with reference to steps that are presented herein in a certain order, in many instances, these steps can be performed in any order as may be appreciated by one skilled in the art; the novel method is therefore not limited to the particular arrangement of steps disclosed herein.

[0134] Monoclonal antibody 2C7 binds to the lipo-oligosaccharide (LOS) epitope on the surface of live Neisseria gonorrhoeae pathogens. 95% of clinical isolates tested directly from cervical secretions of women in Boston and Nanjing showed the presence of the LOS epitope. Antibody 2C7 could successfully kill these isolates via complement-mediated cell death in a dose-dependent manner. In laboratory7animals, 2C7 was shown to be highly efficacious in mouse vaginal colonization models of Neisseria gonorrhoeae, when delivered intraperitoneally as well as intravenously, in transgenic mice containing FH / C4BP (complement inhibitors that enable gonococci to resist complement-dependent killing), as well as in FH / C4BP transgenic mice co-infected with chlamydia and gonorrhea.Furthermore, strains that are deficient in presence of the LOS epitope are inefficient in colonizing vaginal mucosa in female mice, suggesting a bacterial survival advantage to the presence of LOS epitope.

[0135] To advance 2C7 as a therapeutic candidate, the present inventors have carried out humanization of the molecule. The present inventors have demonstrated the efficacy of humanized antibody constructs in vitro. Humanized 2C7 represents a new paradigm for adjuvant treatment of populations at risk with acute, uncomplicated infection of N. gonorrhoeae and with decreased susceptibility to standard antibiotics, as well as for prevention of re-infection with acute, uncomplicated infection with N. gonorrhoeae, which is fast becoming a growing global health burden.

[0136] The importance of a lactose extension from Hep[2] for expression of 2C7 epitope has been established (see Yamasaki et al.). Detailed elucidation of binding epitope was important to define the specificity of mAb 2C7 and also to establish that such a bacterial epitope does not exist on host tissues.

[0137] Subsequent studies in mice validated the importance of the 2C7 epitope. See Gulati II and Ram I. Unlike any other N. gonorrhoeae LOS epitopes that mimic host glycans - as examples, the lacto-N-neotetraose epitope resembles host paragiobodies and the Gal-Gal-Glc trisaccharide resembles the P' -like blood group antigen - the 2C7 epitope does not resemble any known human glycans.

[0138] In summary, the following considerations make mAb 2C7 an attractive anti-gonococcal immunotherapeutic:1 . LOS is expressed by >95% of isolates in vivo, or minimally passaged isolates - broad strain coverage assured. See Gulati I and Ram I.2. 'Non-self’ epitope - mAb will not react with host tissue.3. Function of the epitope in complement inhibition defined - virulence factor. See Ram S. et al.4. Loss of epitope (e.g., IgtG gene deletion) results in attenuation in mice (colonization defect) (See Gulati I and Ram I) - resistance to vaccine unlikely; will be accompanied by considerable fitness cost.5. Mechanism of action (MoA) of mAb 2C7 in mouse model defined (discussed below). See Gulati S. et al., “Complement alone drives efficacy of a chimeric antigonococcal monoclonal antibody”, PLoS Biol.. 17:e3000323 (2019) (hereinafter "Gulati IIP").6. Mechanistic correlate of protection in mouse model defined (human complement-dependent bactericidal assay). See Gulati III.

[0139] In addition to the unique properties described above, LOS is the most abundant outer membrane component of all N. gonorrhoeae and is important for virulence.

[0140] The present disclosure provides monoclonal antibodies - mAb 2C7 - that specifically bind to gonococcal lipo-oligosaccharide (LOS) as the target antigen, which is abundantly and ubiquitously expressed on the outer membrane by N. gonorrhoeae. The LOS epitope recognized by mAb 2C7 (shaded box in Fig. 1) is expressed by >95% of clinical isolates (see Gulati I and Ram I) and does not mimic host glycan structures. This epitope is important for extended colonization of the genital tract, as demonstrated in studies using the mouse vaginal colonization model (see Gulati II and Ram I). “Escape mutants” that do not bind the vaccine- elicited antibodies or the mAb - for example, by phase-variation “off’ of the IgtG gene (FIG.1 ) - would severely compromise the ability of the gonococcus to survive in its human host, rendering it susceptible to clearance by host innate immunity. Secondly, binding of complement inhibitors (e.g., FH and C4BP) enables gonococci to resist complementdependent killing (see Ram S. et al., “Binding of C4b-binding Protein to Porin: A molecular mechanism of serum resistance of Neisseria gonorrhoeae" , J. Exp. Med., 193:281-96 (2001) (hereinafter “Ram IP”), Ram S. et al. , “Binding of complement factor H to loop 5 of porin protein 1 A: a molecular mechanism of serum resistance of nonsialylated Neisseria gonorrhoeae” , J. Exp. Med., 188:671-80 (1998) (hereinafter "Ram III"). w Ram S. et al., “A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae” , J. Exp. Med., 187:743-52 (1998) (hereinafter “Ram IV”) the disclosures of which are each incorporated herein by reference in their entirety.

[0141] Gonococci bind only to human, but not to non-human FH and C4BP (see Ngampasutadol J. et al., “Human C4b-binding protein selectively interacts with Neisseria gonorrhoeae and results in species-specific infection”. Proc. Natl. Acad. Sci. USA,102: 17142-7 (2005) (hereinafter "Ngampasutadol F), and Ngampasutadol J. et al. , “Human Factor H Interacts Selectively with Neisseria gonorrhoeae and Results in Species-Specific Complement Evasion”, J. Immunol., 180:3426-35 (2008) (hereinafter "Ngampasutadol IF ; the disclosures of which are each hereby incorporated herein by reference in their entirety). Accordingly, gonococci can evade killing by only human, but not non-human complement. Thus, the efficacy of bactericidal antibodies may be overestimated in wild-type mice, whose FH and C4BP do not bind to gonococci.

[0142] Therefore, the present inventors created human FH / C4BP transgenic mice (see Ermert D. et al. , “Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors”, PLoS pathogens, ll:e!005043 (2015), the disclosure of which is hereby incorporated by reference herein in its entirety, to evaluate the efficacy of vaccine antibodies such as mAb 2C7 that rely on a complement for their activity in a ‘human like’ complement milieu.

[0143] The present inventors discovered that the lactose extension from HepII that forms part of the 2C7 epitope (Gal-Glc extension from HepII shown in FIG. 1) can be sialylated (see Ram I) which contributes to virulence by i) decreasing complement activation — e.g., by decreasing activation of complement component C3, and ii) engaging Siglec receptors on immune cells (see Landig C.S. et al., “Evolution of the exclusively human pathogen Neisseria gonorrhoeae'. Human-specific engagement of immunoregulatory”, Siglecs. Evol. Appl., 12:337-49 (2019). the disclosure of which is hereby incorporated by reference herein in its entirety). Both events dampen the host inflammatory response and permit wild type gonococci to survive in vivo. Thus, resistance to mAb 2C7 resulting from loss of 2C7 epitope in LOS (e.g. “escape mutation”) would be associated with a loss in bacterial fitness in vivo.

[0144] Isolated Antigen Binding Polypeptides

[0145] In one aspect, the present invention provides an isolated antigen binding polypeptide that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae, comprising a heavy chain variable region including a human acceptor framew ork region and three non-human complementarity determining regions (CDRs) having sequences Hl, H2. and H3 incorporated into the human chain framew ork region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS(SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3);wherein Xi is N or Q or S or A; X2 is N or Q or S or A; X3 is N or Q or S or A, X4 is W or H or Y or F.

[0146] The Hl sequence may be GYTFTDYNME (SEQ ID NO: 7), the H2 sequence may be VINPNNRFTS (SEQ ID NO: 8), and the H3 sequence may be SRWYQYDY (SEQ ID NO: 9).

[0147] In another aspect, the present invention provides an isolated antigen binding polypeptide that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a light chain variable region including a human acceptor framework region and three non-human complementarity' determining regions (CDRs) having sequences LI, L2, and L3 incorporated into the human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4): the L2 sequence is GDGXrRAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXio (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xe is N or Q or S or A; X7 is N or Q or S or A, Xs is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y.

[0148] The LI sequence may be RSSTGAVTTSNYAN (SEQ ID NO: 10), the L2 sequence may be GINNRAP (SEQ ID NO: 11), and the L3 sequence may be ALWYSNHWV (SEQ ID NO: 12).

[0149] In another aspect, the present invention provides isolated antigen binding polypeptides that bind specifically to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a heavy chain variable region including a first human acceptor framework region and three non-human complementarity' determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the first human acceptor framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS (SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3); wherein Xi is N or Q or S or A; X2 is N or Q or S or A; X3 is N or Q or S or A, X4 is W or H or Y or F, in combination with a light chain variable region including a second human acceptor framework region and three non-human CDRs having sequences LI, L2, and L3 incorporated into the second human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4): the L2 sequence is GIX6X7RAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXio (SEQ ID NO: 6),wherein Xs is N or Q or S or A; XQ is N or Q or S or A; X7 is N or Q or S or A, Xs is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y.

[0150] The first human acceptor framework region and the second human acceptor framework region may be different.

[0151] In each of the above embodiments, the lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae is 2C7.

[0152] The heavy chain variable region may have the amino acid sequence of SEQ ID NO: 13 substituted with at least one amino acid substitution selected from the group consisting of Q1E, V5Q, A9P, V11L, K12V, A16S, V20I, G24A, R38K, A40S, P41H, Q43K, G44S, M48I, A61N, K63N, Q65R, R67K, V68A, M70L, R72V, T74K, T76S, V79A, E82D, S84R, R87T, T91S, Y95F, R98G, and V113L. Preferably, the heavy chain variable region of the amino acid sequence of SEQ ID NO: 13 has at least one amino acid substitution selected from the group consisting of Q1E, V5Q, K12V, V20I, G24A, M48I, V68A, M70L, R72V, T74K, V79A, R98G, and V113L.

[0153] The heavy chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22.

[0154] The light chain variable region may have the amino acid sequence of SEQ ID NO: 25 substituted with at least one amino acid substitution selected from the group consisting of S1Q, S2V,E3V, L4V, D7E, P8S. VIOL, SI IT, V12T, A13S. P14P, Q16E, R19,T, I20L, Y38V, Q40E. G43D, Q44H, A45L. P46F, V47T. L48G, V49L, Y51G. I60V. D62A, S68L, S69I, N71D, T72A, S74A, A82T, D87I, Y89F, and C90F. Preferably, the light chain variable region of SEQ ID NO: 25 has at least one amino acid substitution selected from the group consisting of S2V, L4V, VIOL, V12T, P14P, I20L, Y38V, Q40E, Q44H, P46F, L48G, V49L, Y51G. I60V, S68L, and N71D.

[0155] The light chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-33.

[0156] The heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22, and the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NO: 26-33.

[0157] The isolated antigen binding polypeptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-43.

[0158] The isolated antigen binding polypeptide may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-53.

[0159] Antibody or Antigen binding antibody fragment

[0160] In another aspect, the present invention provides a humanized or chimeric antibody or antigen binding antibody fragment comprising the isolated antigen binding polypeptide of any one of the embodiments described above.

[0161] In one aspect, the present invention provides an antibody or antigen binding antibody fragment thereof that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae, comprising a heavy chain variable region including a human acceptor framework region and three non-human complementarity determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the human chain framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS(SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3); wherein Xi is N or Q or S or A; X2is N or Q or S or A; X3is N or Q or S or A, X4is W or H or Y or E

[0162] The Hl sequence may be GYTFTDYNME (SEQ ID NO: 7), the H2 sequence may be VINPNNRFTS (SEQ ID NO: 8), and the H3 sequence may be SRWYQYDY (SEQ ID NO: 9).

[0163] In another aspect, the present invention provides an antibody or antigen binding antibody fragment thereof that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a light chain variable region including a human acceptor framework region and three non-human complementarity determining regions (CDRs) having sequences LI, L2, and L3 incorporated into the human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4); the L2 sequence is GIX6X7RAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXIO (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xg is N or Q or S or A; X7 is N or Q or S or A, X8is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y.

[0164] The LI sequence may be RSSTGAVTTSNYAN (SEQ ID NO: 10), the L2 sequence may be GINNRAP (SEQ ID NO: 11), and the L3 sequence may be ALWYSNHWV (SEQ ID NO: 12).

[0165] In another aspect, the present invention provides isolated antigen binding polypeptides that binds specifically to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a heavy chain variable region including a first human acceptorframework region and three non-human complementarity determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the first human acceptor framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS (SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3); wherein Xi is N or Q or S or A; X2 is N or Q or S or A; X3 is N or Q or S or A, X4 is W or H or Y or F, in combination with a light chain variable region including a second human acceptor framework region and three non-human CDRs having sequences LI, L2, and L3 incorporated into the second human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4): the L2 sequence is GIX6X7RAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXIO (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xe is N or Q or S or A; X7 is N or Q or S or A, Xs is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y.

[0166] The heavy chain variable region may have the amino acid sequence of SEQ ID NO: 13 substituted with at least one amino acid substitution selected from the group consisting of Q1E, V5Q, A9P, V11L, K12V, A16S, V20I, G24A, R38K, A40S, P41H, Q43K, G44S, M48I, A61N, K63N, Q65R, R67K, V68A, M70L, R72V, T74K, T76S, V79A, E82D, S84R, R87T, T9 IS, Y95F, R98G, and VI 13L. Preferably, the heavy chain variable region of the amino acid sequence of SEQ ID NO: 13 has at least one amino acid substitution selected from the group consisting of Q1E, V5Q, K12V, V20I, G24A, M48I, V68A, M70L, R72V, T74K, V79A, R98G, and V113L.

[0167] The heavy chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22.

[0168] The light chain variable region may have the amino acid sequence of SEQ ID NO: 25 substituted with at least one amino acid substitution selected from the group consisting of S1Q, S2V,E3V. L4V, D7E, P8S, VIOL, S11T, V12T, A13S, P14P, Q16E, R19,T, I20L, Y38V. Q40E, G43D, Q44H, A45L, P46F. V47T, L48G, V49L, Y51G, I60V, D62A, S68L. S69L N71D, T72A, S74A, A82T, D87I, Y89F, and C90F. Preferably, the light chain variable region of SEQ ID NO: 25 has at least one amino acid substitution selected from the group consisting of S2V, L4V, VIOL, V12T, P14P, I20L, Y38V, Q40E, Q44H, P46F, L48G, V49L, Y51G, I60V, S68L, and N71D.

[0169] The light chain variable region may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-33.

[0170] The heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22, and the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NO: 26-33.

[0171] The antibody or antigen binding antibody fragment thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-43.

[0172] The antibody or antigen binding antibody fragment thereof may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-53.

[0173] In one embodiment, the antibody is a humanized antibody or antigen binding antibody fragment having a higher binding affinity to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae in comparison to a binding affinity of a chimeric antibody or antigen binding antibody fragment to the LOS epitope of Neisseria gonorrhoeae.

[0174] In each of the embodiments above, the antibody or antigen binding antibody fragment may be a humanized antibody or antigen binding antibody fragment having a binding affinity to the LOS epitope that is at least 2-4 times greater than the binding affinity of the chimeric antibody or antigen binding antibody fragment to the LOS epitope of Neisseria gonorrhoeae.

[0175] In each of the above embodiments, the LOS epitope of Neisseria gonorrhoeae is 2C7.

[0176] Antibody or Antigen Binding Antibody Fragment with Variant Fc Region

[0177] In yet another aspect, the present invention provides a humanized or chimeric antibody or antigen binding antibody fragment of any one of the embodiments described above having an FcRn binding site in which the Fc region has specific Fc-based mutations to alter the half-life of the antibody or antigen binding antibody fragment in plasma or serum and / or improve effector functions such as activation of complement dependent cytotoxicity (CDC) as described in WO 2017158426, WO2019057564, and Foss, S. et al., “Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria”, Nat. Commun., 15:2007 (2024) (doi. org / 10. 1038 / s41467-024-46321 -9); the disclosures of each of which are hereby incorporated by reference herein in their entirety.

[0178] As described in WO 2017158426 and WO2019057564, the Fc region of IgG includes amino acids from both the CH2 and CH3 domains, and a site on the Fc region mediates interaction with the neonatal Fc receptor (FcRn), which is involved with regulatingthe serum persistence of IgG and albumin, and transports both ligands between different cellular compartments.

[0179] In one embodiment, the humanized or chimeric antibody or antigen binding antibody fragment of any one of the embodiments described above includes an Fc variant having at least one mutation in the Fc region, by which the Fc variant provides altered or improved pH-dependent FcRn binding and extended plasma or serum half-life to the humanized or chimeric antibody or antigen binding antibody fragment; as described in Foss. S. et al. (2024).

[0180] In a further embodiment, the humanized or chimeric antibody or antigen binding antibody fragment of any one of the embodiments described above has at least one mutation in the Fc region which is at one or more positions selected from 311, 428, and 434 as numbered by the Kabat system.

[0181] In a further embodiment, the humanized or chimeric antibody or antigen binding antibody fragment of any one of the embodiments described above has at least one mutation in the Fc region selected from the amino acid substitutions Q311R, M428E, and N434W.

[0182] In still a further embodiment, the humanized or chimeric antibody or antigen binding antibody fragment of any one of the embodiments described above has an Fc region comprising the amino acid substitutions Q311R, M428E, and N434W.

[0183] In still a further embodiment, the humanized or chimeric antibody or antigen binding antibody fragment of any one of the embodiments described above has an Fc region comprising the amino acid sequence of SEQ ID NO: 55.

[0184] Pharmaceutical Composition, Vaccine, and Kit

[0185] In yet another aspect, the present invention provides a pharmaceutical composition comprising the isolated antigen binding polypeptide or the humanized antibody or antigen binding antigen binding antibody fragment described in the paragraphs above, and a pharmaceutically acceptable carrier.

[0186] In one embodiment, a vaccine is provided comprising any one of the isolated antigen binding polypeptide or any one of the humanized antibody or antigen binding antibody fragment described in the paragraphs above.

[0187] In yet another aspect, the present invention provides a kit comprising any one of the isolated antigen binding polypeptides, any one of the humanized antibody or antigen binding antibody fragment thereof, as well as pharmaceutical composition or vaccine as described above, for treatment of a disease such as Neisseria gonorrhoeae. Kits containing an isolatedantigen binding polypeptide or antibodies or antigen binding antibody fragments, of the invention find use in therapeutic or diagnostic assays. The kits further contain instructions on the use thereof or instructions as required by the U.S. Food and Drug Administration for in vitro diagnostic kits.

[0188] In another aspect of the invention, an article of manufacture containing an isolated antigen-binding polypeptide, or an antibody or antigen binding antibody fragment as described herein above, and other materials useful for the treatment, prevention and / or diagnosis of an infection described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and / or diagnosing the infection and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody or antigen binding antibody fragment of the invention. The label or package insert indicates that the composition is used for treating an infection of choice. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat an infection, such as an infection caused by Neisseria gonorrheas. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0189] Methods and Use for Treatment of an Infection

[0190] In another aspect, the present invention provides a method of treating or preventing (immune prophylaxis of) an infection comprising a step of administering any one of the isolated antigen binding polypeptides or any one of the humanized antibody or antigen binding antibody fragment thereof described above. In addition, a pharmaceutical composition or a vaccine comprising any one of the isolated antigen binding polypeptides, or any one of the humanized antibodies or antigen binding antibody fragments thereof, as described herein above, may be administered to a patient with the infection.

[0191] In another aspect, the present invention provides use of any one of the isolated antigen binding polypeptides, or any one of the humanized antibody or antigen binding antibody fragment, as well as use of a pharmaceutical composition or vaccine of paragraph comprising any one of the isolated antigen binding polypeptides, or any one of the humanized antibody or antigen binding antibody fragment, for the treatment or preventing (immune- prophylaxis) of an infection. The infection may be a sexually transmitted infection, or an infection caused by Neisseria gonorrhoeae.

[0192] Method of Making Isolated Antigen Binding Polypeptides and Antibodies or Antigen binding antibody fragments

[0193] In another aspect, the present invention provides a method of making any one of the isolated antigen binding polypeptides or antibody and antigen binding antibody fragments thereof described above, comprising introducing a non-human complementarity determining region (CDR) into a human framework region.EXAMPLES

[0194] TABLE A- EXEMPLARY ANTIBODIES

[0195] EXAMPLE 1 - Humanized Antibody Sequences for 2C7 Antibody The VH and VL sequences were compared to a library' of know n human germline sequences from human VH genes and human VLkappa genes (IMGT® the internationalImMunoGeneTics information system® found dot imgt.org; founder and director:Marie-Paule Lefranc, Montpellier, France). ; the databases used were IMGT human VH genes (F+ORF) and IMGT human VLkappa genes (F+ORF) as used by the NCBI IgBLAST program. The acceptor human germline was chosen from those closest in sequence to the parental antibody.

[0196] For VH, human germline IGHVl-46(allele 1) was chosen as the acceptor sequence and the human heavy chain IGHJ6(allele 1 ) joining region (J gene) was chosen from human joining region sequences compiled at IMGT® the international ImMunoGeneTics information system® www.imgt.org (see FIG. 15).

[0197] For VL, human germline IGLV3-19 (allele 1) was chosen as the acceptor sequence and human light chain IGKJ2(allele 1) joining region (J gene) was chosen from human joining region sequences compiled a IMGT® the international ImMunoGeneTics information system® wwwdot imgt.org (see Figure 16).

[0198] CDRs were defined according to the AbM definition (see the website at www dot bioinf.org.uk / abs / for a table comparing CDR definitions).

[0199] Alteration of human germline framework (i.e., non-CDR residues in VH and VL) positions to corresponding parental murine sequence might be required to optimize binding of the humanized antibody. Sites for modification in each humanized sequence are noted in FIGS. 15 and 16.

[0200] In the VH domain, Asn33 and Asn52 (sequential numbering) have low potential for deamidation based on sequence and conformation; Asn54 and Asn55 have medium potential for deamidation. TrplOl is likely partially solvent- exposed and could undergo oxidation under stress conditions. In the VH domain, QI may also be altered to El (Q1E) to prevent pyroGlu formation.

[0201] In the VL domain, Asn33 and Asn96 (sequential numbering) have low potential for deamidation based on sequence and conformation; Asn54 and Asn55 have medium potential for deamidation based on sequence and conformation. Trp93 and Trp98 may undergo oxidation under stress conditions.

[0202] TABLE 1 shows the H2C7 VH-VL pairs.

[0203] TABLE 1

[0204] EXAMPLE 2 - Mouse Infection Studies

[0205] The mouse vaginal colonization model of gonorrhea was used, as described in Jerse II, cited hereinabove. Female wild-type BALB / c mice (The Jackson Laboratory ) in the diestrus phase of the estrous cycle were started on treatment (that day) with 0.5 mg Premarin (Pfizer) in 200 pL water given subcutaneously on each of 3 days; -2, 0, and +2 days (before, the day of, and after inoculation) to prolong the estrus phase of the cycle and promote susceptibility to N. gonorrhoeae infection. Premarin is a mixture of sodium estrone sulfate and sodium equilin sulfate and as concomitant components, sodium sulfate conjugates of 17a-dihydroequilin, 17a-estradiol. and 17P-dihydroequilin. Antibiotics (vancomycin, colistin, neomycin, trimethoprim, and streptomycin) ineffective against N. gonorrhoeae used in challenge experiments were used to reduce competitive microflora.

[0206] Mice were challenged intravaginally with the indicated inoculum of N. gonorrhoeae. as previously described Gulati II, as cited herein above. Infection was monitored daily through vaginal swabbing and bacterial enumeration (CFUs). A dual / competitive infection experiment was performed using a mixture of FA1090wt and FA I ()90 / g / G' (constructed with a tetracycline-resistance marker) to determine if the 2C7 LOS structure provided gonococci a survival advantage. Female BALB / c mice 5-6 wks. of age (Jackson Laboratories) in the diestrus phase of the estrous cycle were treated with water soluble 17|3-estradiol (Sigma) and antibiotics as described previously in Jerse II anA the following day were vaginally inoculated with a mixture of FA1090wt (1.5 105CFU) andFA 1090 / / G (1.6xio5CFU) on Day 0 and colony forming counts (CFUs) performed daily by duplicate plating. 100 pl vaginal swab rinses were quantitatively cultured for N. gonorrhoeae daily (43) onto GC agar supplemented with vancomycin, colistin, nystatin, trimethoprim and streptomycin (GC-VCNTS) and GC-VCNTS plus tetracycline (0.2 pg / ml); the latter permitted growth of FA1090 lgtG~ but not the wt strain. Three characteristics of the data were measured as described in Gulati II. time to clearance, longitudinal trends in mean logio CFU. and the cumulative CFU as AUC. Median time to clearance was estimated using Kaplan-Meier survival curves; times to clearance were compared between groups using the Mantel-Cox log-rank test. The mean AUC (logio CFU) was computed for each mouse to estimate the bacterial burden over time (cumulative infection); the means under the curves were compared between groups using Mann Whitneys test.

[0207] EXAMPLE 3 - Characterization of Cell Culture Supernatants Containing H2C7 mAbs 1-8

[0208] Western Blotting

[0209] Tissue culture supernatants (5 pL) containing each of the 8 humanized 2C7 mAbs were added to 4x LDS sample buffer (Invitrogen) and separated on a 4-12% Bis-Tris gel. Proteins were transferred to a PVDF membrane by western blotting. Blots were blocked with PBS / 1% dry non-fat milk for 1 h and then probed with anti-human IgG (y-chain specific) alkaline phosphatase conjugate (1: 1000 in PBS / 0.05% Tween 20) for 1 h at 37 °C and developed with BCIP / NBT (5-Bromo-4-chloro-3-indonyl phosphate I Nitro-blue- tetrazolium chloride) substrate. As shown in FIG. 6, purified chimeric mAb 2C7 were also run as controls (amounts indicated at the top of the respective lanes).

[0210] EXAMPLE 4 - Binding of H2C7 mAbs 1-8 to Bacterial Lysates

[0211] Binding of humanized mAb 2C7s (H2C7-1 through H2C7-8) to bacterial lysates by ELISA. Lysates of MS 11 isogenic mutant strains that varied in LOS structure (4-Hex / G+ and 2-Hex / G+, both express the 2C7 epitope (as shown in TABLE 2 below) while 4-Hex / G- and 2-Hex / G- are 2C7-negative. A detailed description of these MS 11 isogenic mutants can be found in Chakraborti et al., J. Immunol., 196(11)4576-86 (2016) (PMID: 27183633), the disclosure of which is herein incorporated by reference in its entirety). MS 11 isogenic mutants were coated onto microtiter wells for 2 hours at 37°C. See FIGS. 7A-7I.

[0212] Bacteria were suspended in PBS to an OD 600mn of 0.4 and frozen at -20 °C.Bacterial suspensions were thawed and the lysed cells were coated onto microtiter wells for 2h at 37°C. Wells were blocked with PBS / 0.05% Tween 20 for 1 h at room temperature, followed by the addition of tissue culture supernatants containing the humanized mAbs at the various dilutions indicated on the X-axis. After incubation for 1 h at 37°C, bound IgG was disclosed with either anti-human IgG (Sigma) or anti-human IgG (y-chain specific) alkaline phosphatase conjugate (Promega), followed by the addition of PNPP substrate. See FIGS. 7A-7I.

[0213] TABLE 2

[0214] EXAMPLE 5- Binding of H2C7 mAbs 1-8 to CP2 Biotin

[0215] Binding of humanized mAb 2C7s to a peptide mimic of the 2C7 LOS epitope. A biotinylated cyclized peptide mimic of the 2C7 oligosacchande epitope, called CP2-biotin, which was dissolved in PBS / 0.05% Tween 20 (CP2-biotin concentration was 5 pg / rnL) and captured on Streptavidin coated microtiter plates (ThermoFisher) for 2 hours at room temperature. Following incubation for 2 h at room temperature, wells were washed thrice with PBS / 0.05% Tween 20 and incubated with the tissue culture supernatants containing the humanized 2C7 molecules (either undiluted (neat) or at a 1 / 10 dilution in PBS / TWEEN 20) for 1 h at 37°C. Chimeric human mAh 2C7 was also included at concentrations of either 10 pg / ml (black bar indicated by asterisk *) or 1 pg / ml (grey bar indicated by no asterisk). Bound IgG was disclosed with anti-human IgG (y-chain specific) alkaline phosphatase conjugate (Sigma) or anti-human IgG (y-chain specific) alkaline phosphatase conjugate (Promega), followed by PNPP substrate. See FIG. 8.

[0216] EXAMPLE 6 - Binding of H2C7 mAb Supernatants to MS11 LOS Mutants

[0217] Binding of humanized mAb 2C7s (H2C7-1 through H2C7-8) supernatants to MS11 isogenic mutant strains are shown in FIGS. 9A-9F.

[0218] Binding of humanized mAb 2C7s (H2C7-1 through H2C7-8) to bacterial lysates by ELISA. Lysates of MSI 1 isogenic mutant strains that varied in LOS structure (4-Hex / G+ and 2-Hex / G+, was performed in a manner similar to Example 3 which both express the 2C7 epitope) were coated onto microtiter wells for 2 hours at 37°C. Wells were blocked with PBS / 0.05% Tween 20 for 1 h at room temperature, followed by the addition of tissue culture supernatants containing the humanized mAbs at the various dilutions indicated on the X-axis. After incubation for 1 hour at 37°C, bound IgG was disclosed with anti-human IgG (y-chain specific) alkaline phosphatase conjugate (Promega) and PNPP substrate.

[0219] H2C7-1 to H2C7-5 was tested on Plate 1, and H2C7-6 to H2C7-8 and chimeric mAh 2C7 were tested on Plate 2. A secondary conjugate (anti-human IgG (H + L), Alkaline Phosphatase Conjugate (Promega, Cat No S382B) was the control. The horizontal dashed (at OD of 0.08) represents the conjugate control in FIGS. 9A-9F.

[0220] At a 1 / 10 dilution, none of the H2C7 mAbs bound the 4Hex / G- and 2Hex / G- (both 2C7 neg) - all ODs <0.16.

[0221] EXAMPLE 7 - Binding of H2C7 mAbs 1-8 to MS 4Hex / G+

[0222] Binding of humanized mAb 2C7s (H2C7-1 through H2C7-8) supernatants to MS 11 4Hex / G+ mutants are shown in FIG. 10.

[0223] Binding of humanized mAb 2C7s (H2C7-1 through H2C7-8) to bacterial lysates by ELISA was performed in a manner similar to Example 3. Lysates of strain MS 11 4-Hex / G+, which expresses the 2C7 epitope, was coated onto microtiter wells for 2 hours at 37°C. Wells were blocked with PBS / 0.05% Tween 20 for 1 hour at room temperature, followed by the addition of tissue culture supernatants containing the humanized mAbs at the various dilutions indicated on the X-axis. After incubation for 1 hour at 37°C, bound IgG was disclosed with anti-human IgG (y-chain specific) alkaline phosphatase conjugate (Promega) and PNPP substrate.

[0224] EXAMPLE 8 - Quantitation of H2C7 in Supernatants and Binding Activity of H2C7 mAbs

[0225] Binding activity of H2C7 mAbs and chim 2C7 to MS 11 4-Hex / G+ were quantitated as show n in TABLE 13 below. The concentration of mAbs in the tissue culture supernatants was estimated by ELISA using a standard curve generated with human IVIg (Gammagard).The X-axis of FIG. 12 indicates the concentration of mAh in the supernatants, and the Y-axis of FIG. 12 shows the absorbance at 405 nm at 30 min.

[0226] TABLE 3 shows the quantity of H2C7 in supernatants and binding to 4Hex / G+

[0227] EXAMPLE 9 - Bactericidal Activity of H2C7 mAbs 6-8

[0228] Serum bactericidal activity of purified humanized mAbs H2C7-6, H2C7-7 and H2C7- 8 versus N. gonorrhoeae 15253. N. gonorrhoeae strain 15253 (-1000 CFU) was incubated with the humanized mAbs or chimeric mAb 2C7 ("Chim. 2C7”) at the concentrations indicated on the X-axis. Normal human serum (final concentration 20% (v / v / )) was used as a complement source. The volume of the reaction mixture was maintained at 75 pL. Duplicate aliquots of 15 pL were plated on chocolate agar at time 0 min and 30 min. Plates were incubated at 37 °C in an atmosphere containing 5% CO2. The number of colonies surviving at 30 min relative to CFUs at 0 min was expressed as % survival (Y-axis) in FIG. 12.

[0229] EXAMPLE 10 - Bactericidal Activity of H2C7 mAbs 8-11 against Gonococci

[0230] Serum bactericidal assays (SBAs) were performed using described methods in Gulati S. et al., "'Complement-Dependent Serum Bactericidal Assays for Neisseria gonorrhoeae” , Methods Mol. Biol., 1997:267-80 (2019); the disclosure of which is herein incorporated by reference. Briefly, N. gonorrhoeae strains 15253 or FA1090 (-1000 CFU) were incubated with the humanized mAbs or chimeric mAb 2C7 at the concentrations indicated for each experiment. Normal human serum (final concentration 20% (v / v / )) or IgG and IgM depleted normal human serum (Pel-Freez) were used as complement sources. The volume of the reaction mixture was maintained at 75 pL. Duplicate aliquots of 15 pL were plated on chocolate agar at times 0 min and 30 min. Plates were incubated at 37 °C in an atmosphere containing 5% CO2. The number of colonies surviving at 30 min relative to CFUs at 0 min was expressed as % survival (Y-axis) in FIGS. 13A-13B.

[0231] The IC50 values corresponding to FIG. 13A are shown in TABLE 4, and the IC50 values corresponding to FIG. 13B are shown in TABLE 5.TABLE 4TABLE 5

[0232] EXAMPLE 11 - Bactericidal Activity of H2C7-8 against Gonococci

[0233] Serum bactericidal assays (SBAs) were performed using methods as described in Example 10. N. gonorrhoeae strains FA1090 and 15253 (-1000 CFU) were incubated with humanized H2C7-8 with wild-type (WT) IgGl Fc or humanized H2C7-8 with a derivative containing mutant IgGl Fc (triple REW mutations; hereinafter “REW-Fc” or “Fc-REW”) designed to enhance complement activation, and 20% human complement (IgG and IgMdepleted normal human serum; Pel-Freez), at the concentrations indicated on the x-axis in FIGs. 19A-19B for each experiment. The final reaction volume contained -1000 CFU of N. gonorrhoeae, and the final reaction volume was 50 uL. Aliquots of 5 pL were plated onto chocolate agar at t=0 min and again at t=30 min following incubation of the mixture at 37 °C. Plates were incubated for 24 h at 37 °C and CFUs surviving at 30 min relative to 0 min, expressed as a percentage, are shown on the Y-axis of FIGS. 19A-19B. The IC50 (calculated concentration expected to result in 50% bacterial survival) for the H2C7-8 molecule with REW Fc is indicated. The IC50 for the H2C7-8 molecule with WT IgGl Fc could not be accurately defined.

[0234] The REW-Fc of the H2C7-8 is shown in FIG. 20 with three amino acid substitutionsQ311R / M428E / N434W (REW) as described in WO2017158426A1 and has enhanced plasma half-life and mucosal distribution as described in Foss, S. et al., “Human IgG Fc-engineering for enhanced plasma half-life, mucosal distribution and killing of cancer cells and bacteria”, Nat. Commun., 15:2007 (2024) (https: / / doi.org / 10.1038 / s41467-024-46321-9). As described in WO2019057564, an antibody comprising an REW-Fc is understood to possess a high affinity to human FcRn at pH 5.5-6 (a dissociation equilibrium constant (KD) of less than 100 nM) and have no affinity or a low affinity to human FcRn at pH 7.4 in comparison to the affinity to human FcRn at pH 5.5-6.

Claims

CLAIMS1. An isolated antigen binding polypeptide that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a heavy chain variable region including a human framework region and three non-human complementarity determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the human framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS(SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3); wherein Xi is N or Q or S or A; X2is N or Q or S or A; X3is N or Q or S or A, X4is W or H or Y or F.

2. An isolated antigen binding polypeptide that specifically binds to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a light chain variable region including a human framework region and three non-human complementarity determining regions (CDRs) having sequences LI, L2, and L3 incorporated into the human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4); the L2 sequence is GIX6X7RAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXIO (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xe is N or Q or S or A; X7 is N or Q or S or A, X8is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y.

3. An isolated antigen binding polypeptide that binds specifically to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae comprising a heavy chain variable region including a first human framework region and three non-human complementarity determining regions (CDRs) having sequences Hl, H2, and H3 incorporated into the first human framework region, wherein: the Hl sequence is GYTFTDYXiME (SEQ ID NO: 1); the H2 sequence is VINPX2X3RFTS (SEQ ID NO: 2); and the H3 sequence is SRX4YQYDY (SEQ ID NO: 3); wherein Xi is N or Q or S or A; X2is N or Q or S or A; X3is N or Q or S or A, X4is W or H or Y or F, in combination with a light chain variable region including a second human light chainframework region and three non-human CDRs having sequences LI, L2, and L3 incorporated into the second human framework region, wherein: the LI sequence is RSSTGAVTTSX5YAN (SEQ ID NO: 4); the L2 sequence is GIXeXyRAP (SEQ ID NO: 5); and the L3 sequence is ALWYX8NX9WXIO (SEQ ID NO: 6), wherein X5 is N or Q or S or A; Xe is N or Q or S or A; X7 is N or Q or S or A, Xs is S or H or F or Y; X9 is H or Q or S or A; and X10 is V or H or F or Y.

4. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the Hl sequence is GYTFTDYNME (SEQ ID NO: 7).

5. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the H2 sequence is VINPNNRFTS (SEQ ID NO: 8).

6. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the H3 sequence is SRWYQYDY (SEQ ID NO: 9).

7. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the LI sequence is RSSTGAVTTSNYAN (SEQ ID NO: 10).

8. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the L2 sequence is GINNRAP (SEQ ID NO: 11).

9. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the L3 sequence is ALWYSNHWV (SEQ ID NO: 12).

10. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the human framework region outside of the Hl, H2, and H3 sequences in the heavy chain variable region is an amino acid sequence of SEQ ID NO: 13 which has at least one amino acid substitution selected from the group consisting of Q1E, V5Q, A9P, VI IL, K12V, A16S, V20I, G24A, R38K, A40S, P41H, Q43K, G44S, M48I, A61N, K63N, Q65R, R67K, V68A, M70L, R72V, T74K, T76S, V79A, E82D, S84R, R87T, T9 IS, Y95F, R98G, and V113L.

11. The isolated antigen binding polypeptide of claim 10, wherein the at least one amino acid substitution is selected from the group consisting Q1E, V5Q, K12V, V20I, G24A, M48I, V68A, M70L, R72V, T74K, V79A, R98G, and V113L.

12. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22.

13. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the human framework region outside of the LI, L2, and L3 sequences in the light chain variable region is an amino acid sequence of SEQ ID NO: 25 having at least one amino acid substitution selected from the group consisting of S1Q, S2V,E3V, L4V, D7E, P8S, VIOL, S11T, V12T, A13S, P14P, Q16E, R19,T, I20L, Y38V, Q40E, G43D, Q44H, A45L, P46F, V47T, L48G, V49L, Y51G, I60V, D62A, S68L, S69I, N71D, T72A, S74A, A82T, D87I, Y89F, and C90F.

14. The isolated antigen binding polypeptide of claim 13, wherein the at least one amino acid substitution is selected from the group consisting of S2V, L4V, VIOL, V12T, P14P, I20L, Y38V, Q40E, Q44H, P46F, L48G, V49L, Y51G, I60V, S68L, and N71D.

15. The isolated antigen binding polypeptide of any one of claims 1-3, wherein the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-33.

16. The isolated antigen binding polypeptide of claim 3, wherein the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-20 and 22, and the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NO: 26-33.

17. The isolated antigen binding polypeptide of claim 1, wherein the heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 36-43.

18. The isolated antigen binding polypeptide of claim 2, wherein the light chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NOs: 45-53.

19. The isolated antigen binding polypeptide of claim 3, wherein the first human framework region and the second human framework are different.

20. A humanized antibody or antigen binding antibody fragment comprising the isolated antigen binding polypeptide of any one of claims 1-19.

21. The humanized antibody or antigen binding antibody fragment of claim 20, having a higher binding affinity to a lipo-oligosaccharide (LOS) epitope of Neisseria gonorrhoeae in comparison to a binding affinity of a chimeric antibody or antigen binding antibody fragment to the LOS epitope.

22. The humanized antibody or antigen binding antibody fragment of claim 21 , wherein the humanized antibody or antigen binding antibody fragment has a binding affinity to the LOS epitope that is at least 2-4 times greater than the binding affinity of the chimeric antibody or antigen binding antibody fragment to the LOS epitope.

23. The humanized antibody or antigen binding fragment of any one of claims 20-22, further comprising at least one mutation in an Fc region and having an altered binding affinity for FcRn.

24. The humanized antibody or antigen binding fragment of claim 23, wherein the at least one mutation in the Fc region is at one or more positions selected from the group consisting of 311, 428, and 434, using the Kabat numbering system.

25. The humanized antibody or antigen binding fragment of any one of claims 23-24, wherein the at least one mutation in the Fc region is selected from the group consisting of Q311R, M428E, and N434W, using the Kabat numbering system.

26. The humanized antibody or antigen binding fragment of any one of claims 23-25, wherein the Fc region comprises the amino acid substitutions Q3 HR, M428E, and N434W, using the Kabat numbering system.

27. The humanized antibody or antigen binding fragment of claim 26, wherein the Fc region comprises the amino acid sequence of SEQ ID NO: 55.

28. A pharmaceutical composition comprising the isolated antigen binding polypeptide of any one of claims 1-19, or the humanized antibody or antigen binding antibody fragment of any one of claims 20-27; and a pharmaceutically acceptable carrier.

29. A vaccine comprising the pharmaceutical composition of claim 28.

30. A method of treating an infection comprising a step of administering the isolated antigen binding polypeptide of any one of claims 1-19, or the humanized antibody or antigen binding antibody fragment of any one of claims 20-27, or the pharmaceutical composition of claim 28, or the vaccine of claim 29, to a patient with the infection.

31. The method of claim 30, wherein the infection is an infection caused by Neisseria gonorrhoeae .

32. Use of the isolated antigen binding polypeptide of any one of claims 1-19, or the humanized antibody or antigen binding antibody fragment of any one of claims 20-27, or the pharmaceutical composition of claim 28, or the vaccine of claim 29, for the treatment of an infection.

33. The use of claim 32, wherein the infection is an infection caused by Neisseria gonorrhoeae .

34. A kit for treatment said kit comprising the isolated antigen binding polypeptide of any one of claims 1-19, the humanized antibody or antigen binding antibody fragment of any one of claims 20-27, the pharmaceutical composition of claim 28, or the vaccine of claim 29, and instructions for using the isolated antigen binding polypeptide, the humanized antibody orantigen binding antibody fragment, the pharmaceutical composition, or the vaccine, for treatment of an infection.

35. The kit of claim 34, wherein the infection is an infection caused by Neisseria gonorrhoeae.

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