Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

In vivo affinity maturation of the CD4 domains of an HIV-1-entry inhibitor

Nature Biomedical Engineering volume 8, pages 1715–1729 (2024)Cite this article

Subjects

Abstract

Human proteins repurposed as biologics for clinical use have been engineered through in vitro techniques that improve the affinity of the biologics for their ligands. However, the techniques do not select against properties, such as protease sensitivity or self-reactivity, that impair the biologics’ clinical efficacy. Here we show that the B-cell receptors of primary murine B cells can be engineered to affinity mature in vivo the human CD4 domains of the HIV-1-entry inhibitor CD4 immunoadhesin (CD4-Ig). Specifically, we introduced genes encoding the CD4 domains 1 and 2 (D1D2) of a half-life-enhanced form of CD4-Ig (CD4-Ig-v0) into the heavy-chain loci of murine B cells and adoptively transferred these cells into wild-type mice. After immunization, the B cells proliferated, class switched, affinity matured and produced D1D2-presenting antibodies. Somatic hypermutations in the D1D2-encoding region of the engrafted cells improved the binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein and the inhibitor’s ability to neutralize a panel of HIV-1 isolates without impairing its pharmacokinetic properties. In vivo affinity maturation of non-antibody protein biologics may guide the development of more effective therapeutics.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Engineering primary murine B cells to express a B-cell receptor with CD4 domains 1 and 2.
Fig. 2: Engineered B cells generate neutralizing responses in immunized mice.
Fig. 3: Engineered B cells persist in vivo following immunization.
Fig. 4: D1D2-expressing B cells hypermutate and class switch in vivo.
Fig. 5: Diverse and convergent amino acid mutations in engrafted mice.
Fig. 6: In vivo hypermutations in D1D2 improve the neutralization potency of CD4-Ig-v0.
Fig. 7: CD4-Ig variants bind Env trimers with higher affinity than CD4-Ig-v0.

Similar content being viewed by others

Data availability

Sequences of CD4 inserts at mouse IgH loci are available from the NCBI Sequence Read Archive (SRA) via the accession code PRJNA1124571. The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data for the figures are provided with this paper.

Code availability

Dandelions and dsa source code and binary installers can be downloaded from Github at https://github.com/baileych-bi/dandelions (ref. 56) and https://github.com/baileych-bi/dsa-win64 (ref. 57).

References

  1. Ebrahimi, S. B. & Samanta, D. Engineering protein-based therapeutics through structural and chemical design. Nat. Commun. 14, 2411 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Beerli, R. R. et al. Isolation of human monoclonal antibodies by mammalian cell display. Proc. Natl Acad. Sci. USA 105, 14336–14341 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Boder, E. & Wittrup, D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

    Article  CAS  PubMed  Google Scholar 

  4. Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317 (1985).

    Article  CAS  PubMed  Google Scholar 

  5. Yin, Y. et al. In vitro affinity maturation of broader and more-potent variants of the HIV-1–neutralizing antibody CAP256-VRC26.25. Proc. Natl Acad. Sci. USA 118, e2106203118 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ren, X., Zhao, M., Lash, B., Martino, M. M. & Julier, Z. Growth factor engineering strategies for regenerative medicine applications. Front. Bioeng. Biotechnol. 7, 469 (2019).

    Article  PubMed  Google Scholar 

  7. Haynes, B. F. et al. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308, 1906–1908 (2005).

    Article  CAS  PubMed  Google Scholar 

  8. Chen, J. et al. Mechanism of HIV-1 neutralization by antibodies targeting a membrane-proximal region of gp41. J. Virol. 88, 1249–1258 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Arnold, D. P., Xu, Y. & Takatori, S. C. Antibody binding reports spatial heterogeneities in cell membrane organization. Nat. Commun. 14, 2884 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bumbaca, D. et al. Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4. mAbs 3, 376–386 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Vugmeyster, Y. et al. In vitro potency, pharmacokinetic profiles and pharmacological activity of optimized anti-IL-21R antibodies in a mouse model of lupus. mAbs 2, 335–346 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Wu, H. et al. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J. Mol. Biol. 368, 652–665 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Victora, G. D. & Nussenzweig, M. C. Germinal centers. Annu. Rev. Immunol. 40, 413–442 (2022).

    Article  CAS  PubMed  Google Scholar 

  14. McHeyzer-Williams, L. J., Milpied, P. J., Okitsu, S. L. & McHeyzer-Williams, M. G. Class-switched memory B cells remodel BCRs within secondary germinal centers. Nat. Immunol. 16, 296–305 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wardemann, H. et al. Predominant autoantibody production by early human B cell precursors. Science 5, 5638 (2003).

    Google Scholar 

  16. von Boehmer, H. & Melchers, F. Checkpoints in lymphocyte development and autoimmune disease. Nat. Immunol. 11, 14–20 (2010).

    Article  Google Scholar 

  17. Dosenovic, P. et al. Anti-HIV-1 B cell responses are dependent on B cell precursor frequency and antigen-binding affinity. Proc. Natl Acad. Sci. USA 115, 4743–4748 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Pae, J., Jacobsen, J. T. & Victora, G. D. Imaging the different timescales of germinal center selection. Immunol. Rev. 306, 234–243 (2022).

    Article  CAS  PubMed  Google Scholar 

  19. He, W. et al. Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates. Immunity 56, 2408–2424.e6 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Yin, Y. et al. In vivo affinity maturation of mouse B cells reprogrammed to express human antibodies. Nat. Biomed. Eng. 8, 361–379 (2024).

    Article  CAS  PubMed  Google Scholar 

  21. Yin, Y. & Farzan, M. Affinity maturation of CRISPR-engineered B cell receptors in vivo. Nat. Biomed. Eng. 8, 341–342 (2024).

    Article  Google Scholar 

  22. Junren, C. et al. Pharmacological activities and mechanisms of hirudin and its derivatives – a review. Front. Pharmacol. 12, 660757 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Daix, T. et al. Intravenously administered interleukin-7 to reverse lymphopenia in patients with septic shock: a double-blind, randomized, placebo-controlled trial. Ann. Intensive Care 13, 17 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Kim, M. Y. et al. A long-acting interleukin-7, rhIL-7-hyFc, enhances CAR T cell expansion, persistence, and anti-tumor activity. Nat. Commun. 13, 3296 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Santagostino, E. et al. Long-acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial. Blood 127, 1761–1769 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bluestone, J. A., St Clair, E. W. & Turka, L. A. CTLA4Ig: bridging the basic immunology with clinical application. Immunity 24, 233–238 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Iglesias, M., Brennan, D. C., Larsen, C. P. & Raimondi, G. Targeting inflammation and immune activation to improve CTLA4-Ig-based modulation of transplant rejection. Front. Immunol. 13, 3296 (2022).

    Article  Google Scholar 

  28. Inoue, M. et al. Bivalent structure of a TNFR2-selective and agonistic TNF-α mutein Fc-fusion protein enhances the expansion activity of regulatory T cells. Sci. Rep. 13, 13762 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Fischer, R., Kontermann, R. E. & Pfizenmaier, K. Selective targeting of TNF receptors as a novel therapeutic approach. Front. Cell Dev. Biol. 8, 401 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Shearer, T. W. et al. Recombinant CD4‐IgG2 in human immunodeficiency virus type 1-infected children: phase 1/2 study. J. Infect. Dis. 182, 1774–1779 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Gardner, M. R. et al. AAV-delivered eCD4-Ig protects rhesus macaques from high-dose SIVmac239 challenges. Sci. Transl. Med. 11, eaau5409 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Fletcher, C. V. et al. Nonlinear pharmacokinetics of high-dose recombinant fusion protein CD4-IgG2 (PRO 542) observed in HIV-1–infected children. J. Allergy Clin. Immunol. 119, 747–750 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Jacobson, M. J. et al. Single‐dose safety, pharmacology, and antiviral activity of the human immunodeficiency virus (HIV) type 1 entry inhibitor PRO 542 in HIV‐infected adults. J. Infect. Dis. 182, 326–329 (2000).

    Article  CAS  PubMed  Google Scholar 

  34. Gardner, M. & Farzan, M. Cd4 muteins and methods of using the same. US patent 20230159616-A1 (2023).

  35. Fetzer, I. et al. eCD4-Ig variants that more potently neutralize HIV-1. J. Virol. 92, e02011–e02017 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ou, T. et al. Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire. Mol. Ther. 30, 184–197 (2022).

    Article  CAS  PubMed  Google Scholar 

  37. Johnston, C. M., Wood, A. L., Bolland, D. J. & Corcoran, A. E. Complete sequence assembly and characterization of the C57BL/6 mouse Ig heavy chain V region. J. Immunol. 176, 4221–4234 (2006).

    Article  CAS  PubMed  Google Scholar 

  38. Lin, S. G. et al. Highly sensitive and unbiased approach for elucidating antibody repertoires. Proc. Natl Acad. Sci. USA 113, 7846–7851 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Abbott, R. K. et al. Precursor frequency and affinity determine B cell competitive fitness in germinal centers, tested with germline-targeting HIV vaccine immunogens. Immunity 48, 133–146.e6 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Gardner, M. R. et al. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges. Nature 519, 87–91 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gardner, M. R. & Farzan, M. Engineering antibody-like inhibitors to prevent and treat HIV-1 infection. Curr. Opin. HIV AIDS 12, 294–301 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Rogozin, I. B. & Diaz, M. Cutting edge: DGYW/WRCH is a better predictor of mutability at G:C bases in Ig hypermutation than the widely accepted RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. J. Immunol. 172, 3382–3384 (2004).

    Article  CAS  PubMed  Google Scholar 

  43. de Carvalho, R. V. H. et al. Clonal replacement sustains long-lived germinal centers primed by respiratory viruses. Cell 186, 131–146.e13 (2023).

    Article  PubMed  Google Scholar 

  44. Hägglöf, T. et al. Continuous germinal center invasion contributes to the diversity of the immune response. Cell 186, 147–161.e15 (2023).

    Article  PubMed  Google Scholar 

  45. Krykbaev, R., McKeating, J. & Jones, I. Mutant CD4 molecules with improved binding to HIV envelope protein gp120 selected by phage display. Virology 234, 196–202 (1997).

    Article  CAS  PubMed  Google Scholar 

  46. Tsui, P., Sweet, R. W., Sathe, G. & Rosenberg, M. An efficient phage plaque screen for the random mutational analysis of the interaction of HIV-1 gp120 with human CD4. J. Biol. Chem. 267, 9361–9367 (1992).

    Article  CAS  PubMed  Google Scholar 

  47. deCamp, A. et al. Global panel of HIV-1 Env reference strains for standardized assessments of vaccine-elicited neutralizing antibodies. J. Virol. 88, 2489–2507 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Feige, M. J. et al. An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. Mol. Cell 34, 569–579 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Davis-Gardner, M. E., Gardner, M. R., Alfant, B. & Farzan, M. eCD4-Ig promotes ADCC activity of sera from HIV-1-infected patients. PLoS Pathog. 13, e1006786 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Fellinger, C. H. et al. eCD4-Ig limits HIV-1 escape more effectively than CD4-Ig or a broadly neutralizing antibody. J. Virol. 93, e00443-19 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Huang, D. et al. Vaccine elicitation of HIV broadly neutralizing antibodies from engineered B cells. Nat. Commun. 11, 5850 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Nahmad, A. D. et al. Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion. Nat. Commun. 11, 5851 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Lee, J. H. et al. Long-primed germinal centres with enduring affinity maturation and clonal migration. Nature 609, 998–1004 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Sarzotti-Kelsoe, M. et al. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods 409, 131–146 (2014).

    Article  CAS  PubMed  Google Scholar 

  55. Zhu, P., Olson, W. C. & Roux, K. H. Structural flexibility and functional valence of CD4-IgG2 (PRO 542): potential for cross-linking human immunodeficiency virus type 1 envelope spikes. J. Virol. 75, 6682–6686 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bailey C. C. Dandelions: B cell receptor sequence clustering and lineage graph generation. GitHub https://github.com/baileych-bi/dandelions (2024).

  57. Bailey C. C. dsa: Deep Sequencing Analysis. GitHub https://github.com/baileych-bi/dsa-win64 (2024).

Download references

Acknowledgements

We thank H. Peng, N. Elowe and S. Bayani for assisting with SPR experiments and data analysis, and H. Choe for advice and comments on the paper. Funding was provided by the following National Institutes of Health awards to M.F.: U19 AI149646, R21 AI152836, R01 DA056771, UM1 AI126623, R01 AI154989, R37 AI091476 and R01 AI174270.

Author information

Author notes

  1. Wenhui He

    Present address: Institute for Molecular and Cellular Therapy, Chinese Institutes for Medical Research, and School of Basic Medical Sciences, Capital Medical University, Beijing, China

Authors and Affiliations

  1. Skaggs Graduate School, Scripps Research, La Jolla, CA, USA

    Andi Pan, Naomi Bronkema & Michael Farzan

  2. The Center for Integrated Solutions to Infectious Diseases, The Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Andi Pan, Charles C. Bailey, Tianling Ou, Jinge Xu, Xin Liu, Baodan Hu, Naomi Bronkema, Michael Farzan & Wenhui He

  3. Division of Infectious Disease, Boston Children’s Hospital, Boston, MA, USA

    Andi Pan, Tianling Ou, Jinge Xu, Xin Liu, Nickolas Skamangas, Naomi Bronkema, Mai H. Tran, Huihui Mou, Yiming Yin, Michael Farzan & Wenhui He

  4. Department of Pediatrics, Harvard Medical School, Boston, MA, USA

    Andi Pan, Tianling Ou, Jinge Xu, Xin Liu, Nickolas Skamangas, Naomi Bronkema, Mai H. Tran, Huihui Mou, Yiming Yin, Michael Farzan & Wenhui He

  5. Center for the Development of Therapeutics, The Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Tonia Aristotelous

  6. The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology, Jupiter, FL, USA

    Gogce Crynen

  7. The Scripps Research Institute, Jupiter, FL, USA

    Xia Zhang

  8. Emmune Inc., Cambridge, MA, USA

    Michael D. Alpert

Authors
  1. Andi Pan
    View author publications

    Search author on:PubMed Google Scholar

  2. Charles C. Bailey
    View author publications

    Search author on:PubMed Google Scholar

  3. Tianling Ou
    View author publications

    Search author on:PubMed Google Scholar

  4. Jinge Xu
    View author publications

    Search author on:PubMed Google Scholar

  5. Tonia Aristotelous
    View author publications

    Search author on:PubMed Google Scholar

  6. Xin Liu
    View author publications

    Search author on:PubMed Google Scholar

  7. Baodan Hu
    View author publications

    Search author on:PubMed Google Scholar

  8. Gogce Crynen
    View author publications

    Search author on:PubMed Google Scholar

  9. Nickolas Skamangas
    View author publications

    Search author on:PubMed Google Scholar

  10. Naomi Bronkema
    View author publications

    Search author on:PubMed Google Scholar

  11. Mai H. Tran
    View author publications

    Search author on:PubMed Google Scholar

  12. Huihui Mou
    View author publications

    Search author on:PubMed Google Scholar

  13. Xia Zhang
    View author publications

    Search author on:PubMed Google Scholar

  14. Michael D. Alpert
    View author publications

    Search author on:PubMed Google Scholar

  15. Yiming Yin
    View author publications

    Search author on:PubMed Google Scholar

  16. Michael Farzan
    View author publications

    Search author on:PubMed Google Scholar

  17. Wenhui He
    View author publications

    Search author on:PubMed Google Scholar

Contributions

A.P., M.F. and W.H. conceived the study and designed experiments. A.P., W.H., T.O., J.X., X.Z., N.S. and T.A. performed and analysed experiments. A.P., C.C.B. and B.H. designed and performed bioinformatic analysis. C.C.B. developed software tools. W.H., T.O., J.X., X.L., M.H.T., N.B., H.M., M.D.A. and Y.Y. developed key reagents and provided useful insight. A.P. and G.C. conducted statistical analyses. M.F. provided funding support. A.P., M.F. and W.H. wrote the paper.

Corresponding authors

Correspondence to Michael Farzan or Wenhui He.

Ethics declarations

Competing interests

A.P., W.H., T.O., Y.Y. and M.F. are inventors of a patent describing the in vivo affinity maturation of antibodies and biologics. C.C.B., M.D.A. and M.F. have equity stakes in Emmune, Inc., which developed CD4-Ig-v0. The other authors declare no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks Paula Cannon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Strategies for presenting human biologics on the BCR of primary murine B cells.

a Designs of CD4 D1D2-presenting B-cell receptors. Below each structure is a representation of the cassette used to generate it. D1D2 (yellow) was attached to the N terminus of OKT3 heavy-chain variable region (CD4-OKT-VH, green, left) via a (G4S)3 linker or attached directly to OKT3 kappa light-chain constant region (light blue, centre and right). This light-chain constant region was linked to that of the heavy chain via a (G4S)3 linker (CD4-Cĸ-GS, centre), or non-covalently associated with heavy-chain constant region by P2A cleavage (CD4-Cĸ-P2A, right). b The editing strategy used to express these constructs in primary murine B cells. A gRNA targets an intronic region immediately downstream of the J4 segment. Repair template homology arms complement this region, facilitating insertion of a cassette with a poly-A tail, a splice acceptor (SA), a heavy-chain promoter, a D1D2 construct, and a splice donor (SD). Although this editing strategy is efficient, it interferes with affinity maturation of the expressed B-cell receptor20. An alternate strategy, used hereafter, is represented in Fig. 1b. c Flow cytometric analysis of singlet viable B cells edited using the strategy in b were stained with anti-IgM antibodies and either anti-human CD4 antibodies or HIV-1 gp120 at 48 h post-editing. d Expression of multiple biologics on primary mouse B cells. Using the editing strategy presented in Fig. 1b, OKT-VH fusions of human SIRPα, CTLA-4, and IL-7 were introduced into the heavy-chain locus of primary murine B cells. HDRT were delivered with AAV-DJ. Cells were analyzed 48 h post-electroporation by flow cytometry for their ability to bind human CD47, human CD80, or anti-IL-7 antibodies. Note that CTLA-4 and IL-7 have murine receptors that can bind the secreted antibody fusion to recognize both edited and unedited cells. a, b are created with BioRender.com.

Supplementary information

Source data

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pan, A., Bailey, C.C., Ou, T. et al. In vivo affinity maturation of the CD4 domains of an HIV-1-entry inhibitor. Nat. Biomed. Eng 8, 1715–1729 (2024). https://doi.org/10.1038/s41551-024-01289-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41551-024-01289-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research