Accurate prediction of the structurally diverse complementarity determining region heavy chain 3 (CDR-H3) loop structure remains a primary and long-standing challenge for antibody modeling. Here, we present the H3-OPT toolkit for predicting the 3D structures of monoclonal antibodies and nanobodies. H3-OPT combines the strengths of AlphaFold2 with a pre-trained protein language model and provides a 2.24 Å average RMSDCα between predicted and experimentally determined CDR-H3 loops, thus outperforming other current computational methods in our non-redundant high-quality dataset. The model was validated by experimentally solving three structures of anti-VEGF nanobodies predicted by H3-OPT. We examined the potential applications of H3-OPT through analyzing antibody surface properties and antibody-antigen interactions. This structural prediction tool can be used to optimize antibody-antigen binding and engineer therapeutic antibodies with biophysical properties for specialized drug administration route.

The naïve human antibody repertoire has theoretical access to an estimated > 1015 antibodies. Identifying subsets of this prohibitively large space where therapeutically relevant antibodies may be found is useful for development of these agents. It was previously demonstrated that, despite the immense sequence space, different individuals can produce the same antibodies. It was also shown that therapeutic antibodies, which typically follow seemingly unnatural development processes, can arise independently naturally. To check for biases in how the sequence space is explored, we data mined public repositories to identify 220 bioprojects with a combined seven billion reads. Of these, we created a subset of human bioprojects that we make available as the AbNGS database (https://naturalantibody.com/ngs/). AbNGS contains 135 bioprojects with four billion productive human heavy variable region sequences and 385 million unique complementarity-determining region (CDR)-H3s. We find that 270,000 (0.07% of 385 million) unique CDR-H3s are highly public in that they occur in at least five of 135 bioprojects. Of 700 unique therapeutic CDR-H3, a total of 6% has direct matches in the small set of 270,000. This observation extends to a match between CDR-H3 and V-gene call as well. Thus, the subspace of shared (\'public\') CDR-H3s shows utility for serving as a starting point for therapeutic antibody design.

Antibodies, through their ability to target virtually any epitope, play a key role in driving the adaptive immune response in jawed vertebrates. The binding domains of standard antibodies are their variable light (VL) and heavy (VH) domains, both of which present analogous complementarity-determining region (CDR) loops. It has long been known that the VH CDRs contribute more heavily to the antigen-binding surface (paratope), with the CDR-H3 loop providing a major modality for the generation of diverse paratopes. Here, we provide evidence for an additional role of the VL domain as a modulator of CDR-H3 structure, using a diverse set of antibody crystal structures and a large set of molecular dynamics simulations. We show that specific attributes of the VL domain such as subtypes, CDR canonical forms and genes can influence the structural diversity of the CDR-H3 loop, and provide a physical model for how this effect occurs through inter-loop contacts and packing of CDRs against each other. Our results indicate that the rigid minor loops fine-tune the structure of CDR-H3, thereby contributing to the generation of surfaces complementary to the vast number of possible epitope topologies, and provide insights into the interdependent nature of CDR conformations, an understanding of which is important for the rational antibody design process.

The immune systems protect vertebrates from foreign molecules or antigens, and antibodies are important mediators of this system. The sequences and structural features of antibodies vary depending on species. Many of antibodies from vertebrates, including camelids, have both heavy and light chain variable domains, but camelids also have antibodies that lack the light chains. In antibodies that lack light chains, the C-terminal variable region is called the VHH domain. Antibodies recognize antigens through six complementarity-determining regions (CDRs). The third CDR of the heavy chain (CDR-H3) is at the center of the antigen-binding site and is diverse in terms of sequence and structure. Due to the importance of antibodies in basic science as well as in medical applications, there have been many studies of CDR-H3s of antibodies that possess both light and heavy chains. However, nature of CDR-H3s of single-domain VHH antibodies is less well studied. In this chapter, we describe current knowledge of sequence-structure-function correlations of single-domain VHH antibodies with emphasis on CDR-H3. Based on the 370 crystal structures in the Protein Data Bank, we also attempt structural classification of CDR-H3 in single-domain VHH antibodies and discuss lessons learned from the ever-increasing number of the structures.

Therapeutic antibody discovery using synthetic diversity has been proved productive, especially for target proteins not suitable for traditional animal immunization-based antibody discovery approaches. Recently, many lines of evidences suggest that the quality of synthetic diversity design limits the development success of synthetic antibody hits. The aim of our study is to understand the quality limitation and to properly address the challenges with a better design. Using VH3-23 as a model framework, we observed and quantitatively mapped CDR-H3 loop length-dependent usage of human IGHJ4 and IGHJ6 germline genes in the natural human immune repertoire. Skewed usage of DH2-JH6 and DH3-JH6 rearrangements was quantitatively determined in a CDR-H3 length-dependent manner in natural human antibodies with long CDR-H3 loops. Structural modeling suggests choices of JH help to stabilize antibody CDR-H3 loop and JH only partially contributes to the paratope. Our observations shed light on the design of next-generation synthetic diversity with improved probability of success.

Covalent target modulation with small molecules has been emerging as a promising strategy for drug discovery. However, covalent inhibitory antibody remains unexplored due to the lack of efficient strategies to engineer antibody with desired bioactivity. Herein, we developed an intracellular selection method to generate covalent inhibitory antibody against human rhinovirus 14 (HRV14) 3C protease through unnatural amino acid mutagenesis along the heavy chain complementarity-determining region 3 (CDR-H3). A library of antibody mutants was thus constructed and screened in vivo through co-expression with the target protease. Using this screening strategy, six covalent antibodies with proximity-enabled bioactivity were identified, which were shown to covalently target HRV14-3C protease with high inhibitory potency and exquisite selectivity. Compared to structure-based rational design, this library-based screening method provides a simple and efficient way for the discovery and engineering of covalent antibody for enzyme inhibition.

Although at first glance the diversity of the immunoglobulin repertoire appears random, there are a number of mechanisms that act to constrain diversity. For example, key mechanisms controlling the diversity of the third complementarity determining region of the immunoglobulin heavy chain (CDR-H3) include natural selection of germline diversity (DH ) gene segment sequence and somatic selection upon passage through successive B-cell developmental checkpoints. To test the role of DH gene segment sequence, we generated a panel of mice limited to the use of a single germline or frameshifted DH gene segment. Specific individual amino acids within core DH gene segment sequence heavily influenced the absolute numbers of developing and mature B-cell subsets, antibody production, epitope recognition, protection against pathogen challenge, and susceptibility to the production of autoreactive antibodies. At the tip of the antigen-binding loop (PDB position 101) in CDR-H3, both natural (germline) and somatic selection favored tyrosine while disfavoring the presence of hydrophobic amino acids. Enrichment for arginine in CDR-H3 appeared to broaden recognition of epitopes of varying hydrophobicity, but led to diminished binding intensity and an increased likelihood of generating potentially pathogenic dsDNA-binding autoreactive antibodies. The phenotype of altering the sequence of the DH was recessive for T-independent antibody production, but dominant for T-cell-dependent responses. Our work suggests that the antibody repertoire is structured, with the sequence of individual DH selected by evolution to preferentially generate an apparently preferred category of antigen-binding sites. The result of this structured approach appears to be a repertoire that has been adapted, or optimized, to produce protective antibodies for a wide range of pathogen epitopes while reducing the likelihood of generating autoreactive specificities.

Sequential developmental checkpoints are used to \"optimize\" the B cell antigen receptor repertoire by minimizing production of autoreactive or useless immunoglobulins and enriching for potentially protective antibodies. The first and apparently most impactful checkpoint requires μHC to form a functional pre-B cell receptor (preBCR) by associating with surrogate light chain, which is composed of VpreB and λ5. Absence of any of the preBCR components causes a block in B cell development that is characterized by severe immature B cell lymphopenia. Previously, we showed that preBCR controls the amino acid content of the third complementary determining region of the H chain (CDR-H3) by using a VpreB amino acid motif (RDR) to select for tyrosine at CDR-H3 position 101 (Y101). In antibodies bound to antigen, Y101 is commonly in direct contact with the antigen, thus preBCR selection impacts the antigen binding characteristics of the repertoire. In this work, we sought to determine the forces that shape the peripheral B cell repertoire when it is denied preBCR selection. Using bromodeoxyuridine incorporation and evaluation of apoptosis, we found that in the absence of preBCR there is increased turnover of B cells due to increased apoptosis. CDR-H3 sequencing revealed that this is accompanied by adjustments to DH identity, DH reading frame, JH, and CDR-H3 amino acid content. These adjustments in the periphery led to wild-type levels of CDR-H3 Y101 content among transitional (T1), mature recirculating, and marginal zone B cells. However, peripheral selection proved incomplete, with failure to restore Y101 levels in follicular B cells and increased production of dsDNA-binding IgM antibodies.

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, DH reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD(+), memory IgD(-), and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse, the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.

In directed evolution experiments, a single randomization scheme of an antibody gene does not provide optimal diversity for recognition of all sizes of antigens. In this study, we have expanded the recognition potential of our universal library, termed ScFvP, with a second distinct diversification scheme. In the second library, termed ScFvM, diversity was designed closer to the center of the antigen binding site in the same antibody framework as earlier. Also, the CDR-H3 loop structures were redesigned to be shorter, 5-12 aa and mostly without the canonical salt bridge between Arg106H and Asp116H to increase the flexibility of the loop and to allow more space in the center of the paratope for binding smaller targets. Antibodies were selected from the two libraries against various antigens separately and as a mixture. The origin and characteristics of the retrieved antibodies indicate that complementary diversity results in complementary functionality widening the spectrum of targets amenable for selection.