Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Here, we report the isolation and characterization of high-affinity, human CFH monoclonal antibodies (mAbs) from single B cells of patients with the autoantibody. Using our lead candidate mAb, we explore the mechanism of anti-tumor cell activity and demonstrate the ability of the antibody to kill tumor cells in vitro, and inhibit tumor growth in vivo.

In order to develop a CFH antibody as a cancer therapeutic, targeting the same epitope that is recognized by the autoantibodies of cancer patients would be essential to prevent off-target effects, since CFH is a ubiquitous protein that binds to the surface of host cells. CFH is a multifunctional 150 kDa protein that is composed of 20 short consensus repeats (SCRs), each 60 amino acids long (). The C-terminal SCR domains SCR19 and SCR20 bind to glycosaminoglycan and sialic acid polyanions, as well as to membrane-bound C3b and its proteolytic fragments, on the mammalian cell surface (). CFH antibodies affinity purified from the sera of lung cancer patients bind an epitope within SCR19, PIDNGDIT (). This epitope comprises residues that are predicted on mutational and structural grounds to be critical for the CFH-C3b interaction (). In vitro, the CFH antibodies prevent CFH binding to tumor cells, increase C3b binding, and promote complement-dependent cytotoxicity (CDC). Although CFH is abundant in the blood, it is notable that patients who have these antibodies show no apparent off-target effects. This, and the fact that the autoantibodies bind preferentially to reduced over oxidized CFH in vitro, led us to propose that the autoantibodies bind to a conformationally distinct form of CFH that exists in tumors ().

CFH is a regulatory protein that protects host cells from attack and destruction by the complement system by inhibiting the alternative pathway of complement-mediated lysis (). CFH prevents the deposition of complement C3b on the cell surface by several mechanisms. Deposition of C3b initiates the formation of cell-lytic membrane attack complexes (MACs) leading to cell lysis. Thus, CFH inhibition of the deposition of C3b on the cell surface protects against cell lysis. Tumor cells take advantage of the protection conferred by CFH in order to evade destruction by the complement system (). We hypothesize that by neutralizing this protective protein, patient antibodies to CFH allow complement activation and tumor cell lysis, resulting in the release of anaphylatoxins and modulation of the adaptive immune response, thus suppressing tumor growth while forestalling metastasis.

Metastatic disease is responsible for the majority of cancer deaths, and unfortunately, many current drugs only offer modest benefits in progression-free survival. We have been studying the immune response in a distinct group of patients with early-stage disease who do not develop metastasis as an approach to developing therapeutic strategies (). Our goal was to identify tumor-specific antibodies capable of initiating tumor cell death while stimulating a durable, long-term adaptive immune response. We previously reported an association of autoantibodies to a complement regulatory protein, complement factor H (CFH), with early-stage non-small cell lung cancer (NSCLC) and found that patients with stage I NSCLC had a significantly higher incidence of anti-CFH antibody than those with late-stage NSCLC (p = 0.0051). This association led to the hypothesis that CFH antibodies that arise in lung cancer patients may promote anti-tumor cell activity and that CFH antibody administration might provide a unique way to stimulate a long-term immune response and treat cancer. We set out to isolate and characterize human CFH antibodies starting from the memory B cells of patients with the autoantibody in an effort to develop a therapy that would recapitulate the native immune response.

In order to test antibody efficacy in a mouse with a functional immune system, we used the KLN205-DBA/2 syngeneic lung cancer model (). The murine KLN205 cell line expresses CFH and binds murine mAb7968 (data not shown). Tumor cells were injected subcutaneously (s.c.), then mAb7968 or negative control mAbNctl was injected intraperitoneally (i.p.) on days 1, 4, 7, 10, and 13. Tumor volumes were measured periodically thereafter. Differences in mean tumor volume were observed in the two groups of mice, with systemically administered mAb7968 conferring growth delay and inhibition compared to negative control mAbNctl ( Figure 5 A). The magnitude of this difference reached statistical significance (p < 0.05). H&E staining of a section from the residual tumor from a mAb7968-treated mouse showed an abundant lymphocytic infiltrate that was absent in the tumor section from a control mouse ( Figure 5 B).

(A) Growth curves. KLN205 tumor cells were injected s.c. on day 0, and mAb7968 or mAbNctl was injected i.p. every 3 days between days 1 and 13, after which treatment was stopped. The mean tumor volumes ± SEM for t = 7 mice treated with each mAb are plotted. p values for the difference in tumor volumes between mAb7968 and mAbNctl on days 39, 42, and 45 are 0.027, 0.030, and 0.077, respectively.

For in vivo mouse studies, we developed a murine version of mAb7968 since initial experiments had shown that the human mAb triggered the formation of anti-human antibodies in nude mice. To test the effect of antibody on tumor growth, we initially used an adult-patient-derived brain tumor xenograft, D-270MG, grown in nude mice (), performing intratumoral injections of either murine IgG1-mAb7968, a murine subtype-matched negative control antibody, or no antibody in each of three groups of mice. Injections were repeated biweekly for 3 weeks, and tumors were measured. By the end of the 3-week study, there was significant tumor growth inhibition ( Figures S3 A and S3B) and prolonged survival ( Figure S3 C) in the group of animals that received murine mAb7968. The primary concern for side effects from inhibition of CFH by a CFH antibody is renal toxicity (). Stained sections from the kidneys of all animals were examined by H&E and were normal. There were no observed adverse reactions at necropsy in any of the animals treated with mAb7968. H&E-stained sections of tumor excised from mice receiving the negative control mAb show densely packed tumor cells, whereas H&E-stained sections from the smallest palpable mass excised from an mAb7968-treated mouse show diffuse inflammatory cells without visible tumor cells ( Figure S3 D).

To confirm complement activation, we assayed products of the CDC reaction (C3a, C5a, and C5b-9). The anaphylatoxins C3a and C5a are generated during complement activation and as cytokines for immune cells are links between the innate and adaptive immune systems (). C5b-9 comprises the terminal MAC, a minimal number of which are required to assemble in order to lyse the cell (). Addition of mAb7968 to A549 or H226 cells in the presence of NHS resulted in increased C3a release ( Figure 4 A), C5a release ( Figure 4 B), and C5b-9 deposition ( Figure 4 C).

(C) C5b-9 deposition on A549 or H226 cells. After incubation with NHS, NHS plus mAb7968, or NHS plus a subtype-matched negative control mAb (Neg), C5b-9 deposition on cells was measured by flow cytometry.

(B) Release of C5a from A549 or H226 cells. Using the same cell supernatants described in (A), C5a was measured by ELISA.

(A) Release of C3a from A549 or H226 lung cancer cells. Cells were incubated in the presence of NHS alone, NHS plus mAb7968, or NHS plus human IgG. C3a was measured in cell supernatants at 1 hr and 4 hr by ELISA.

Because of the location of the epitope within the CFH protein and inferred mechanism of action, we tested five of the mAbs in a CDC assay using A549 lung cancer cells. Lung cancer cells were chosen because we originally discovered CFH autoantibodies in lung cancer patients (). In the CDC assay, cells are mixed with antibodies and normal human serum (NHS) as a source of complement and incubated at 37°C, and cytolysis is measured by lactate dehydrogenase (LDH) release. The five mAbs behaved similarly in the CDC assay; however, we chose mAb7968 for future development because, in addition to its low-nanomolar affinity for the epitope-containing peptide, it was highly expressed in cell culture. When added to A549 cells, mAb7968 increased CDC over that seen with a negative control human antibody by 101% ( Figure 3 ). Besides A549 (a NSCLC adenocarcinoma cell line), other cell lines that were killed by mAb7968 were the H226 squamous cell carcinoma and H460 large cell cancer cell lines (both NSCLC cell lines), the DMS79 small cell lung cancer cell line, the SKBR3 breast cancer cell line, and the KATOIII gastric cancer cell line ( Figure 3 ). Additional cell lines that showed increased cytotoxicity with mAb7968 were A431 (epidermoid carcinoma), RD-ES (Ewing’s sarcoma), and FaDu (pharyngeal squamous cell carcinoma) (data not shown). All cell lines were shown to express CFH and bind mAb7968 (data not shown).

Importantly, the peptide exhibited a conformation distinctly different from that observed in other structures of CFH protein constructs (). In particular, residues 1,117–1,120 (sequence element NGDI) near the C terminus of the peptide adopted an α-helical conformation in the antibody-bound complex, whereas the same region exhibited a β strand conformation in natively folded structures of CFH ( Figure 2 A) (). This was consistent with the observation that mAb7968 bound a conformationally distinct form of CFH ( Figure S1 ). The presence of the helical element was also consistent with the Ala scanning results showing a discontinuous epitope for mAb7968 ( Figure S2 ).

Asp1119 in the peptide made an H-bond with Thr56 on the heavy chain at a location on the surface of the complex. Disruption of this interaction by substitution with Ala resulted in varying impact on antibody binding ( Figure S2 ). Thus with this structure, disrupting a through-water interaction (Asn1117) located on the interior of the antibody-antigen interface was more detrimental to binding than disrupting a superficial direct interaction (Asp1119).

To investigate the interaction of the CFH antibody with its epitope, the crystal structure of Fab7968 in complex with a CFHpeptide was solved by molecular replacement and refined to a resolution of 2.0 Å ( Figure 2 A; Table S2 ). Most of the CFH polypeptide backbone (residues 1,112–1,122) was visible in the scattering electron density, showing specific antibody-antigen interactions in detail. In particular, the CFH residues Asn1117 and Ile1120 had been shown by alanine scanning to be critical components of the mAb7968 epitope ( Figure S2 ). The complex structure showed that Asn1117 made a through-water interaction with heavy chain residue Gly96. It is also possible that the substitution of Asn1117 with Ala disrupted an intra-peptide interaction between the Asn1117 and Thr1121 side chains that helped to stabilize the peptide’s α-helical conformation. In agreement with this, the Thr1121Ala mutation partially but not entirely abrogated binding ( Figure S2 ). The complex structure also showed the Ile1120 side chain binding in a hydrophobic pocket formed by Val50 and Tyr58 on the heavy chain and Trp96 on the light chain. Mutating Ile1120 to Ala would allow solvent to penetrate this pocket, disrupting hydrophobic antibody-antigen contacts in the vicinity.

(B) CFH peptide in its antibody-bound conformation (red) contrasted in a superposition to the same region in the natively folded CFH protein (gray). Here, only the 7,968 epitope is highlighted in red (antibody-bound conformation) and yellow (on the natively folded protein).

(A) Fab 7968 shown with the heavy chain in green and the light chain in blue. The paratope with bound peptide in red is oriented toward the top of the figure, and the CDRs are marked and labeled.

To define the amino acid footprint of the recombinant CFH mAbs, we analyzed binding of seven recombinant mAbs to alanine-substituted peptides containing the originally described 8-amino-acid binding domain ( Figure S2 ). Mutation of Ile1120 to Ala completely ablated binding of all mAbs; mutation of Asn1117 resulted in 0%–35% of wild-type binding, and mutation of Thr1121 result in 1%–66% of wild-type binding. None of the mAbs were affected by changes in the four residues upstream or three residues downstream of the epitope that were included in the peptide.

Most of the VDJand Vsequences in the individual clonal lineages had identical sequences except the fourth group in Table 1 . Seven antibodies with unique VDJand Vsequences were selected and produced as purified recombinant antibodies for further characterization. These antibodies were demonstrated to bind CFH with specificity for the reduced form of both full-length CFH and a fragment containing SCR19-20 ( Figure S1 ). Thus, the isolated antibodies recapitulate the specificity of the autoantibodies identified in sera. By surface plasmon resonance analysis (SPR), six of the seven antibodies had affinities in the low-nanomolar range against the CFH epitope-peptide ( Table S1 ). Taken together, these data confirm that single human B cells against this tumor antigen express high-affinity antibodies that have undergone somatic hypermutation.

We had previously mapped the binding region of the autoantibodies from NSCLC patient sera to a specific 8-amino-acid segment (PIDNGDIT) of the SCR19 domain (). In order to clone human mAbs that recognize this epitope, we pooled the peripheral blood mononuclear cells (PBMCs) of 11 patients who were shown by immunoblot to express CFH autoantibody. Using a biotinylated CFH 15-mer peptide containing the mapped 8-amino-acid binding segment as bait, we sorted single memory B cells from the pooled PBMCs by flow cytometry for isolation of the immunoglobulin (Ig) variable region of heavy and light gene segments (VDJand V) ( Figure 1 A). VDJand Vgene pairs were isolated by RT/PCR from each single CFH antigen-specific memory B cell. A total of 15 recombinant mAbs generated from the isolated VDJand Vgene pairs were identified that bound to the peptide containing the epitope ( Figure 1 B). Gene sequence analysis indicated that these 15 CFH-positive mAbs belong to seven clonal lineages ( Table 1 ). Although these lineages used different Vgene families, all used a kappa light chain with a CDR3 of 9 amino acids in length. The antibodies had Vgene mutation frequencies ranging from 2.7% to 10.9% and Vchain mutation frequencies ranging from 3.0% to 6.3%, except that both VDJand Vgenes of Ab7966 had 0% mutation. Except for two immunoglobulin M (IgM) antibodies (Ab7962 and Ab7966), these antibodies were IgG3 subtype, as were all 22 autoantibodies previously subtyped in NSCLC patient sera ().

Antibodies were regarded as clonally related if they were inferred to use the same V and J gene segments, had identical CDR3 length, and had 70% or greater nucleotide identity in the CDR3 region, while D-segment identification is subject to substantial uncertainty, so inferred differences in D-segment use were not sufficient to rule out clonal relatedness of two antibodies. Clonally related antibodies are grouped. Based on the analysis, seven antibodies (in bold) with unique V H DJ H and V L J L sequences were selected to be produced as purified antibodies for further characterization.

(B) Identification of cloned mAbs that bind a CFH epitope-containing peptide by ELISA. Supernatants from HEK293 cells transiently expressing pairs of V H and V L chains were collected, adjusted to 1 μg/mL IgG, and equal volumes tested for binding to epitope peptide bound to neutravidin (Pep-NA) or to neutravidin alone (NA). Bound antibody was detected with anti-human IgGγ-HRP and ABTS/H 2 O 2 substrate. Sample 31 is an affinity purified anti-CFH autoantibody serving as a positive control.

(A) Isolation of CFH-specific memory B cells that bind to the epitope of interest. PBMCs were obtained from patients positive for anti-CFH antibodies and used for sorting CFH antigen-binding memory B cells via fluorescence-activated cell sorting. To minimize false positives, streptavidin was labeled with Alexa Fluor 647 and Brilliant Violet 421. Labeling with each fluorophore was carried out on separate aliquots of streptavidin, which were then mixed together prior to interaction with biotinylated antigen peptide (CFH 1110-1124 ). Cells showing elevated fluorescence for both fluorophores, indicated by the outlined region (0.32% of the total), were sorted into single wells for recombinant mAb synthesis. The B cell expressing mAb7968 is designated in pink.

Discussion

Morris et al., 2011 Morris L.

Chen X.

Alam M.

Tomaras G.

Zhang R.

Marshall D.J.

Chen B.

Parks R.

Foulger A.

Jaeger F.

et al. Isolation of a human anti-HIV gp41 membrane proximal region neutralizing antibody by antigen-specific single B cell sorting. In an effort to develop an immunotherapeutic strategy, we initially embarked on a search for autoantibodies associated with a distinct non-metastatic early-stage phenotype that could cause cancer cell death, modulate the adaptive immune response, and ultimately produce a long-term cellular response against the tumor. The current study used a unique approach to develop a tumor-specific antibody that would target cancer cells without creating off-target effects. Here, we report the sequencing and expression of CFH antibodies starting from the B cells of patients who produced these antibodies. While this same technology has been used to isolate broadly neutralizing antibodies for HIV starting from B cells (), this study isolates high-affinity antibodies with anti-tumor cell and anti-tumor growth activity directly from patients. The process of cloning and expressing antibody genes derived from selected B cells is significantly more efficient than production of mAbs in mice by immunization followed by “humanization.” This allowed us to generate an affinity matured antibody that recognizes a conformationally distinct epitope of CFH that, when originally targeted by the immune system, resulted in a desirable phenotype (i.e., limitation of early stage cancer and no apparent side effects).

The 15 isolated CFH-reactive antibodies can be classified into seven clonal lineages because they share the same V H , J H , V κ and J κ gene families and had the same HCDR3 and KCDR3 lengths. Since the PBMCs that were used for sorting single B cells were pooled from 11 patients, it is unclear if antibody members from the individual clonal lineages were from one patient or from different patients.

Campa et al., 2015 Campa M.J.

Gottlin E.B.

Bushey R.T.

Patz Jr., E.F. Complement factor H antibodies from lung cancer patients induce complement dependent lysis of tumor cells, suggesting a novel immunotherapeutic strategy. The CFH mAbs have the same specificity for the conformationally distinct form of CFH and the SCR19-20 fragment as the serum autoantibodies previously described (), which is important to avoid potential off-target effects. An altered conformation of the CFH epitope is seen in the peptide-antibody co-crystal structure, and recognition of this conformation in the tumor environment may be the basis for antibody tumor specificity. This finding also suggests that conformationally distinct epitopes may be relevant therapeutic targets.

We showed that CFH mAb7968 killed a variety of tumor cell lines in vitro, including NSCLC (three histological types), small cell lung cancer, gastric cancer, and breast cancer. In vivo, growth of both a subcutaneous brain tumor in a nude mouse model and a subcutaneous lung tumor in a syngeneic model were substantially inhibited by this antibody without observable adverse reactions.