Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Alterations in thymocyte surface markers after in vivo treatment by serum thymic factor.

Three T cell markers (heterologous sheep rosette, autologous rosette, and theta antigen) have been studied in thymocytes after in vivo injection of a single dose of serum thymic factor (Facteur Thymique Sérique: FTS). Within 24 h after such treatment, sheep erythrocyte-binding cells increased 5 to 8 times, while autologous rosette-forming cells decreased by a factor of 3. Using cytotoxicity assays (trypan blue exclusion test and chromium release) it was observed that thymocytes from FTS-treated mice presented a higher sensitivity to antitheta serum and complement than control mice. These results are compatible with the hypothesis that exposure of immature T cells to thymic hormone may induce T cell maturation, as assessed by decreased number of immature autologous rosette forming cells (RFC) and increased number of mature T cells (sheep RFC and theta-positive cells). Facteur Thymique Sérique appears as an interesting probe to define the various intra-thymic cellular compartments of differentiation.     

Immune cytolysis of human malignant melanoma by antibody to oncofetal antigen I (OFA-I)

Summary Oncofetal antigen I (OFA-I) has been identified by immunofluorescence and immune adherence (IA) as a membrane antigen on human tumor cells, which cross-reacts with fetal brain tissue. This antigen induces humoral antibody in patients with cancer. The present work was designed to evaluate the complement-dependent cytotoxic potential of anti-OFA-I antibody produced in melanoma patients against an OFA-I-positive melanoma cell line, UCLA SO M14 (M14). Patients' sera were chosen on the basis of anti-OFA-I activity by IA. Alloantibodies to M14 were removed by absorption of the sera with lymphoblastoid cells autologous to M14. Fourteen sera were tested, and all demonstrated cytotoxic activity in the presence of rabbit complement. Human complement was also shown to mediate cytotoxicity, although less effectively than rabbit complement. The specificity of the reaction was confirmed when the cytotoxic capability could be absorbed by fetal brain, but not by autologous fetal liver tissues. These results indicate that a patient's serum antibody to OFA-I can lyse tumor cells expressing this antigen.     

Human lymphocyte dependent antibody mediated cytotoxicity: Adherence of lymphocytes to antibody treated target cells

An in vitro human antibody dependent cell mediated cytotoxicity system was used to study the adherence of nonsensitized attacking lymphocytes from peripheral blood to antibody coated melanoma target cells. Specific adherence of attacking cells was documented by labeling the lymphocytes with ⁵¹Cr. The degree of specific adherence was proportional to antibody concentration and incubation time and could be detected before the lysis of target cells. Adsorption of attacking lymphocytes on immune serum treated target cells depleted B cells, enriched T cells, and removed most cytotoxic activity of nonadherent lymphocytes in this system. These results were not found when attacking lymphocytes were adsorbed on normal serum treated target cells.     

Characteristics of macrophage cytotoxicity induced by IgE immune complexes

In earlier studies, the specific adherence of normal rat macrophages to Schistosoma mansoni schistosomula, followed by macrophage cytotoxicity against the larvae, was shown to be induced by incubation of the macrophages with serum from infected rats containing complexes of IgE antibody and circulating schistosome antigens. By the use of a chromium-51 release assay, it is pointed out that this cytotoxic process is a two-step phenomenon. The first step, i.e., activation of normal unstimulated macrophages induced by incubation of the cell with IgE complexes in immune rat serum, is a nonspecific mechanism which may also be elicited by various other macrophage activators. The second step, i.e., immune adherence and cytotoxicity of activated macrophages against S. mansoni schistosomula, is a specific process which imperatively needs the presence of S. mansoni IgE immune complexes. Aggregated myeloma IgE does not activate adherent peritoneal cells into cytotoxic effector cells unless the further participation of these specific IgE immune complexes is provided. The necessary preincubation of macrophages with immune rat serum before adding schistosomula accounts for the inefficiency of the incubation of the target itself with serum to elicit macrophage cytotoxicity. Serum dilution also appears as a critical factor since immune rat serum is inefficient when diluted more than $\text{1}\text{25}$. Aggregated rat IgG neither induces macrophage activation nor inhibits the activation by IgE immune complexes. Though the binding of IgE to the macrophage appears to be isotype specific, homologous immune complexes of IgE antibody and schistosome antigens are required to induce killing of S. mansoni larvae. The possible mechanism of this new model of macrophage activation and cytotoxicity is discussed.     

Serial studies of autologous antibody reactivity to squamous cell carcinoma of the head and neck

Summary In previous studies we evaluated the incidence and specificity of autologous antibody reactivity against squamous cell carcinoma of the head and neck (SCCHN). We were able to demonstrate that autologous antibody reactivity is present in native sera but was usually of too low a titer to allow further analysis. Dissociation of immune complexes by acidification and ultrafiltration of serum augmented autologous antibody reactivity in nine out of nine autologous systems tested. Native antibody and antibody derived from immune complexes produced by the host and reactive with autologous tumor cells may be directed against physiologically relevant antigens. Therefore, correlations of antibody titers with clinical course may provide insight into the nature of the host response to cancer. In the present analysis, serological studies of six patients with SCCHN were performed with serum samples obtained over many months. Results of serial serological assays were correlated to tumor progression and clinical course. Fluctuations in autologous antibody reactivity were noted over time. In four cases, rises in autologous antibody titers preceded the clinical diagnosis of recurrence by several months. Drops in autologous antibody reactivity were noted in two cases following surgery or radiation therapy. In two cases of long-term survivors, no correlation between antibody reactivity and clinical course was noted. Specificity analysis of the six autologous systems demonstrated reactivity against autologous and allogeneic SCCHN as well as melanoma cell lines. These sera did not react with glioma, neuroblastoma, renal cell, breast, bladder and colon carcinoma cell lines nor with fetal calf serum, pooled lymphocytes, red blood cells and platelets. Autologous serial serological studies may provide a means by which to evaluate the host/tumor relationship in patients with SCCHN.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Cytokines produced by blood mononuclear cells stimulated with the streptococcal preparation OK-432: effect on production by supplementing the medium with xenogeneic serum

Summary Human blood MNC were stimulated for 7 days with a streptococcal preparation, OK-432, in a medium supplemented with either 10% autologous serum (autoserum) or 10% FCS. Cytokines, including IL-2, IL-1, IFN, and TNF in the supernatants, and antitumor cytotoxicity of MNC were analyzed. None of the IL-2 was detectable during the culture in medium with autoserum, although significant enhancement of cytotoxicity was observed at day 1. On the other hand, production of IL-2 and higher cytotoxicity were induced in the medium with FCS. Even a control culture of MNC unstimulated with OK-432 in the medium with FCS, showed a slight but significant amount of IL-2 and considerable cytotoxicity. However, such a culture in the medium with autoserum showed no such IL-2 production or cytotoxicity. The cytotoxicity induced in the medium with FCS was significantly inhibited by the addition of anti-IL-2 antibody to the culture as well as by the addition of anti-IFN antibody, whereas the cytotoxicity in the medium with autoserum was not inhibited at all by anti-IL-2 antibody. Therefore, the cytotoxicity induced in the medium with FCS included IL-2-induced killer activity, i.e., LAK activity. Similarly, the levels of IL-1, IFN, and TNF production in the supernatants were variable depending on the serum used to supplement the medium. The nonspecific production of IL-2 and the unexpected induction of cytotoxicity were consistently provoked in the medium using several different lots of FCS, but not in the medium with human allogeneic sera or pooled AB serum nor in that with autoserum. It was revealed that the source of sera used to supplement the medium is an important factor affecting the results of analysis of cytokine production and cytotoxicity of MNC induced by certain stimulants. Abbreviations used: NK, natural killer; IFN, interferon; TNF, tumor necrosis factor; IL-1, interleukin-1; IL-2, interleukin-2; LAK, lymphokine-activated killer; MNC, mononuclear cells; FCS, fetal calf serum; ³H-TdR, ³H-Thymidine     

Blocking of Lymphocyte-mediated Cytotoxicity for Rat Hepatoma Cells by Tumour-specific Antigen-Antibody Complexes

LYMPHOCYTES from tumour-bearing animals are often cytotoxic in vitro against cultured tumour cells from the same individual^1–4. It is possible that the serum of tumour-bearing hosts may contain circulating factors which interfere with the cell-mediated immune responses concerned in tumour rejection reactions⁵. Evidence has been provided by the demonstration that lymphocyte cytotoxicity against cultured tumour cells could be blocked by first exposing tumour cells to serum from tumour-bearing animals^2,3; similar effects have also been observed in cancer patients^6,7. The blocking factor in tumour-bearer serum has the characteristic properties of 7S immunoglobulins², suggesting the involvement of tumour-specific antibody. Serum blocking activity is rapidly lost, however, in animals rendered tumour free and the activity of tumour-bearer serum can be neutralized by the addition of serum from these animals^8,9. One explanation is that the blocking factor in tumour-bearer serum is antigen-antibody complex and the objective of these studies, using a transplanted rat hepatoma (D23), was to test directly whether such complexes prepared from solubilized tumour-specific antigen and antiserum exhibit blocking activity.     

Effector Cell Recruitment with Novel Fv-based Dual-affinity Re-targeting Protein Leads to Potent Tumor Cytolysis and in Vivo B-cell Depletion

Bispecific antibodies capable of redirecting the lytic potential of immune effector cells to kill tumor targets have long been recognized as a potentially potent biological therapeutic intervention. Unfortunately, efforts to produce such molecules have been limited owing to inefficient production and poor stability properties. Here, we describe a novel Fv-derived strategy based on a covalently linked bispecific diabody structure that we term dual-affinity re-targeting (DART). As a model system, we linked an Fv specific for human CD16 (FcγRIII) on effector cells to an Fv specific for mouse or human CD32B (FcγRIIB), a normal B-cell and tumor target antigen. DART proteins were produced at high levels in mammalian cells, retained the binding activity of the respective parental Fv domains as well as bispecific binding, and showed extended storage and serum stability. Functionally, the DART molecules demonstrated extremely potent, dose-dependent cytotoxicity in retargeting human PBMC against B-lymphoma cell lines as well as in mediating autologous B-cell depletion in culture. In vivo studies in mice demonstrated effective B-cell depletion that was dependent on the transgenic expression of both CD16A on the effector cells and CD32B on the B-cell targets. Furthermore, DART proteins showed potent in vivo protective activity in a human Burkitt's lymphoma cell xenograft model. Thus, DART represents a biologically potent format that provides a versatile platform for generating bispecific antibody fragments for redirected killing and, with the selection of appropriate binding partners, applications outside of tumor cell cytotoxicity.     

Cell-mediated cytotoxicity against syngeneic tumors

Spleen cells from DBA/2 mice bearing the DBA/2 P815X mastocytoma for approximately 2 weeks can be stimulated in vitro by mastocytoma cells to generate cytotoxicity measured as ⁵¹Cr release from mastocytoma cells in a 4-hr assay. These cytotoxic cells will not kill allogeneic cell lines but will kill a series of first transplant generation syngeneic tumors. T cells are involved in that treatment of the responding or the cytotoxic cell populations with either anti-T or anti-theta antibody + complement will abrogate all cytotoxicity. Anti-Ly 2.1 antibody + complement treatment of either responder cells (prior to the in vitro culture with irradiated tumor cells) or effector cells after culture markedly decreases cytotoxicity whereas treatment with anti-Ly 1.1 was more effective prior to culture compared to its effect on cytotoxic cells per se. These T cells are in the small lymphocyte class and occur either singly or in aggregates. Suppression of antisyngeneic tumor cytotoxicity by antibody inhibits preferentially the expression of cytotoxicity in the aggregate fractions.     

Irradiated chimeric antigen receptor engineered NK-92MI cells show effective cytotoxicity against CD19+ malignancy in a mouse model

Background aimsAnti-CD19 chimeric antigen receptor (CAR)-modified T cells have shown dramatic cytotoxicity against B-cell malignancies. Currently, autologous T cells are conventionally used to manufacture CAR T cells. Low quality or insufficient quantity of autologous T cells may lead to failure of CAR T preparations. Moreover, CAR T preparation usually takes 1–2 weeks, which is too long for patients with rapid disease progression to successfully infuse CAR T cells. Thus, the development of a ready-to-use CAR immunotherapy strategy is needed. NK-92, a natural killer (NK) cell line derived from an NK lymphoma patient, has been gradually applied as a CAR-modified effector cell. To avoid the potential development of secondary NK lymphoma in patients, large doses of radiation are used to treat NK-92 cells before clinical application, which ensures the safety but reduces the cytotoxicity of NK-92 cells. Therefore, it is crucial to explore a suitable radiation dose that ensures short life span and good cytotoxicity of CAR NK-92 cells.MethodsNK-92MI, a modified IL-2-independent NK-92 cell line, was used to establish an anti-CD19 CAR NK. The suitable radiation dose of CAR NK was then explored in vitro and validated in vivo, and the specific cytotoxicity of irradiated and unirradiated CAR NK against CD19⁺ malignant cells was assessed.ResultsCAR NK exhibited specific cytotoxicity against CD19⁺ malignant cells. Irradiation ensured a short life span of CAR NK in vitro and in vivo. Encouragingly, irradiated CAR NK displayed an anti-CD19⁺ malignancy capacity similar to that of unirradiated CAR NK.ConclusionsFive Gy is a suitable radiation dose to ensure the safety and effectiveness of CD19 CAR NK-92MI cells.     

A Single Domain–Based Anti-Her2 Antibody Has Potent Antitumor Activities

Human epidermal growth factor receptor 2 (HER2) is overexpressed in approximately 20% to 30% of breast cancers and various other types of cancers, which plays a vital role in the cancer progression. Monoclonal antibodies targeting Her2 are now used in the clinic to treat Her2 overexpression cancer patients. However, relapse or resistance is frequent with the current therapies. To generate a new treatment avenue against Her2, we immunized and selected a specific anti-Her2 single domain antibody C3 for further studies. The C3-Fc antibody drove antibody-dependent cell-mediated cytotoxicity against Her2-positive tumor cells in vitro and resulted in potent antitumor growth in vivo. These data suggest that the C3-Fc antibody may provide an alternative avenue for Her2-positive cancer therapy.     

Announcements

Both in vitro sensitization and in vitro expression of a cell-mediated CBA/H anti-DBA/2 response are suppressed by antibody directed against the stimulator or target DBA/2 cells, respectively. Induction of cytotoxicity and the suppression of sensitization or effector phases of the in vitro cell-mediated immune response by antibody are immunologically specific. Specific suppression by antibody-containing serum is (i) highly dependent on the number of stimulating cells used for sensitization, (ii) most marked when antibody is given at the initiation of the sensitization cultures, (iii) more effective in inhibiting the sensitization phase than the effector phase, (iv) interfered with by absorption with cells which contain stimulating or target cell antigens, (v) limited to the 7S fraction of antibody-containing serums, (vi) not markedly dependent on the Fc portion of antibody, and (vii) not augmented, but inhibited slightly, by the formation of antigen-antibody complexes. The characteristics of suppression by antibody in this in vitro system suggest that inhibition occurs mainly through an antigen-masking mechanism and that antibody feedback inactivation of T cells, equivalent to that of B cells, does not take place. The resistance to antibody feedback of T cells involved in the production of cytotoxic effector cells is similar to the resistance of T cells which operate in helper activities in T cell-dependent antibody responses.     

High expression of adhesion molecules/activation markers with little interleukin-2, interferon γ, and tumor necrosis factor β gene activation in fresh tumor-infiltrating lymphocytes from lung adenocarcinoma

Tumor-infiltrating lymphocytes (TIL) were derived from primary breast tumors, metastatic lymph nodes and malignant pleural effusions from 34 patients with breast cancer. TIL were cultured for approximately 30 days and studied for phenotype, cytotoxicity, and the ability to secrete cytokines in response to autologous tumor stimulation. Tumor specimens were obtained from two different sites in 7 patients, resulting in 41 samples from which 38 TIL cultures were established. In addition to screening 38 bulk TIL cultures, TIL from 21 patients were separated into CD4⁺ and CD8⁺ subsets and extensively studied. Three CD4⁺ TIL were found specifically to secrete granulocyte macrophage-colony-stimulating factor and tumor necrosis factor when stimulated by autologous tumor and not by a large panel of stimulators (24–34) consisting of autologous normal cells, allogeneic breast or melanoma tumors and EBV-B cells. This cytokine release was found to be MHC-class-II-restricted, as it was inhibited by the anti-HLA-DR antibody L243. These 3 patients' EBV-B cells, when pulsed with tumor lysates, were unable to act as antigen-presenting cells and induce cytokine secretion by their respective CD4⁺ TIL. These findings demonstrate that MHC-class-II-restricted CD4⁺ T cells recognising tumor-associated antigens can be detected in some breast cancer patients.     

Announcements

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Vaccination of Stage IV patients with allogeneic IL-4- or IL-2-gene-transduced melanoma cells generates functional antibodies against vaccinating and autologous melanoma cells

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.     

ADAM17-overexpressing breast cancer cells selectively targeted by antibody–toxin conjugates

A disintegrin and metalloproteinase 17 (ADAM17) is significantly upregulated not only in malignant cells but also in the pro-inflammatory microenvironment of breast cancer. There, ADAM17 is critically involved in the processing of tumor-promoting proteins. Therefore, ADAM17 appears to be an attractive therapeutic target to address not only tumor cells but also the tumor-promoting environment. In a previous study, we generated a monoclonal anti-ADAM17 antibody (A300E). Although showing no complement-dependent cytotoxicity or antibody-dependent cellular cytotoxicity, the antibody was rapidly internalized by ADAM17-expressing cells and was able to transport a conjugated toxin into target cells. As a result, doxorubicin-coupled A300E or Pseudomonas exotoxin A-loaded A300E was able to kill ADAM17-expressing cells. This effect was strictly dependent on the presence of ADAM17 on the surface of target cells. As a proof of principle, both immunotoxins killed MDA-MB-231 breast cancer cells in an ADAM17-dependent manner. These data suggest that the use of anti-ADAM17 monoclonal antibodies as a carrier might be a promising new strategy for selective anti-cancer drug delivery.     

Isolation and purification of a squamous cell carcinoma of the head and neck-associated antigen identified by autologous antibody

We have previously shown that detection of autologous antibody activity to squamous cell carninoma of the head and neck many be augmented by dissociation in immune complexes. Western blot analysis with autologous antibody has identified a 60 kDa squamous cell carcinoma of the head and neck-associated antigen in spent media and immune complex-dissociated serum ultrafiltrate not recognized by normal human area. Antigen-containing fractions of spent media were eluted from anion exchange columns immediately after serum albumin indicating that the antigen has an acidic PI < 4. Preparative purification of the squamous cell carcinoma antigen was accomplished by anion exchange of concentrated spent media (protein concentration 300 mg/ml) followed by lectin affinity chromotography with a Triticum vulgaris column. A single 60 kDa band was detected by silver stain and Western blot in antigen-containing fractions eluted following lectin affinity chromotography and SDS-PAGE. Final concentration of the antigen was determined to be 1 μm/ml of protein with relative activity increased 1600 × over unfractionated spent media. We conclude that a squamous cell carcinoma of the head and neck-associated antigen, detected by autologous antibody, is an acidic kDa glycoprotein.     

Broadly Neutralizing Antibodies Developed by an HIV-Positive Elite Neutralizer Exact a Replication Fitness Cost on the Contemporaneous Virus

Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1⁺) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.