Activation of ductal progenitor-like cells from adult human pancreas requires extracellular matrix protein signaling.

Ductal progenitor-like cells are a sub-population of ductal cells in the adult human pancreas that have the potential to contribute to regenerative medicine. However, the microenvironmental cues that regulate their activation are poorly understood. Here, we establish a 3-dimensional suspension culture system containing six defined soluble factors in which primary human ductal progenitor-like and ductal non-progenitor cells survive but do not proliferate. Expansion and polarization occur when suspension cells are provided with a low concentration (5% v/v) of Matrigel, a sarcoma cell product enriched in many extracellular matrix (ECM) proteins. Screening of ECM proteins identified that collagen IV can partially recapitulate the effects of Matrigel. Inhibition of integrin α1ß1, a major collagen IV receptor, negates collagen IV- and Matrigel-stimulated effects. These results demonstrate that collagen IV is a key ECM protein that stimulates the expansion and polarization of human ductal progenitor-like and ductal non-progenitor cells via integrin α1ß1 receptor signaling.

Tolerating CD47.

Cluster of differentiation 47 (CD47) occupies the outer membrane of human cells, where it binds to soluble and cell surface receptors on the same and other cells, sculpting their topography and resulting in a pleiotropic receptor-multiligand interaction network. It is a focus of drug development to temper and accentuate CD47-driven immune cell liaisons, although consideration of on-target CD47 effects remain neglected. And yet, a late clinical trial of a CD47-blocking antibody was discontinued, existent trials were restrained, and development of CD47-targeting agents halted by some pharmaceutical companies. At this point, if CD47 can be exploited for clinical advantage remains to be determined. Herein an airing is made of the seemingly conflicting actions of CD47 that reflect its position as a junction connecting receptors and signalling pathways that impact numerous human cell types. Prospects of CD47 boosting and blocking are considered along with potential therapeutic implications for autoimmune diseases and cancer.

Analysis of Circulating Tumor DNA in Synchronous Metastatic Colorectal Cancer at Diagnosis Predicts Overall Patient Survival.

Sporadic colorectal cancer (sCRC) initially presents as metastatic tumors in 25-30% of patients. The 5-year overall survival (OS) in patients with metastatic sCRC is 50%, falling to 10% in patients presenting with synchronous metastatic disease (stage IV). In this study, we systematically analyzed the mutations of RAS, PIK3CA and BRAF genes in circulating tumor DNA (ctDNA) and tumoral tissue DNA (ttDNA) from 51 synchronous metastatic colorectal carcinoma (SMCC) patients by real-time PCR, and their relationship with the clinical, biological and histological features of disease at diagnosis. The highest frequency of mutations detected was in the KRAS gene, in tumor biopsies and plasma samples, followed by mutations of the PIK3CA, NRAS and BRAF genes. Overall, plasma systematically contained those genetic abnormalities observed in the tumor biopsy sample from the same subject, the largest discrepancies detected between the tumor biopsy and plasma from the same patient being for mutations in the KRAS and PIK3CA genes, with concordances of genotyping results between ttDNA and ctDNA at diagnosis of 75% and 84%, respectively. Of the 51 SMCC patients in the study, 25 (49%) showed mutations in at least 1 of the 4 genes analyzed in patient plasma. From the prognostic point of view, the presence and number of the most common mutations in the RAS, PIK3CA and BRAF genes in plasma from SMCC patients are independent prognostic factors for OS. Determination of the mutational status of ctDNA in SMCC could be a key tool for the clinical management of patients.

The TSP1-CD47-SIRPα interactome: an immune triangle for the checkpoint era.

The use of treatments, such as programmed death protein 1 (PD1) or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) antibodies, that loosen the natural checks upon immune cell activity to enhance cancer killing have shifted clinical practice and outcomes for the better. Accordingly, the number of antibodies and engineered proteins that interact with the ligand-receptor components of immune checkpoints continue to increase along with their use. It is tempting to view these molecular pathways simply from an immune inhibitory perspective. But this should be resisted. Checkpoint molecules can have other cardinal functions relevant to the development and use of blocking moieties. Cell receptor CD47 is an example of this. CD47 is found on the surface of all human cells. Within the checkpoint paradigm, non-immune cell CD47 signals through immune cell surface signal regulatory protein alpha (SIRPα) to limit the activity of the latter, the so-called trans signal. Even so, CD47 interacts with other cell surface and soluble molecules to regulate biogas and redox signaling, mitochondria and metabolism, self-renewal factors and multipotency, and blood flow. Further, the pedigree of checkpoint CD47 is more intricate than supposed. High-affinity interaction with soluble thrombospondin-1 (TSP1) and low-affinity interaction with same-cell SIRPα, the so-called cis signal, and non-SIRPα ectodomains on the cell membrane suggests that multiple immune checkpoints converge at and through CD47. Appreciation of this may provide latitude for pathway-specific targeting and intelligent therapeutic effect.

Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata.

Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by ß cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.

Murine Mammary Carcinoma Induces Chronic Systemic Inflammation and Immunosuppression in BALB/c Mice.

PURPOSE: The F3II cell line is a highly invasive variant of mammary carcinoma. Although it is frequently used as a model to evaluate the efficacy of immunotherapy, its impact on the immune system remains poorly understood. The main objectives of this study were to evaluate the effects of F3II tumors on the development of chronic inflammation and to characterize tumor-associated immunosuppression. METHODS: Following the experimental implantation of F3II tumors in BALB/c mice, alterations in the liver and spleen anatomy and the numbers of circulating leukocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells were measured using hematological techniques, histopathological analysis, and flow cytometry. The capacity of the F3II tumor-bearing mice to reject MB16F10 allogeneic tumor transplantation was also evaluated. In addition, the restoration of immune parameters in tumor-bearing mice was evaluated after standard breast cancer chemotherapy and surgical tumor excision. RESULTS: F3II tumor implantation increased the levels of chronic inflammatory markers, such as the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios, and caused myeloid alterations, including extramedullary granulopoiesis and megakaryopoiesis, along with the recruitment of MDSCs to the spleen. Chemotherapy or surgical F3II tumor removal completely rescued the tumor-associated extramedullary granulopoiesis and megakaryopoiesis. Notably, the presence of F3II tumors reduced the capacity of BALB/c mice to reject MB16F10 allogeneic tumor transplantation. CONCLUSION: These results support the occurrence of F3II tumor-mediated immune cell dysfunction, which mimics the immune alterations characterized by chronic systemic inflammation and immunosuppression observed in breast cancer in clinical settings. Thus, the F3II tumor model is relevant for evaluating novel breast cancer immunotherapies and combinations in preclinical studies. This model could also be useful for identifying appropriate therapeutic targets and developing proof-of-concept experiments in the future.

Breaking and restoring immune tolerance to pancreatic beta-cells in type 1 diabetes.

PURPOSE OF REVIEW: Type 1 diabetes (T1D) results from the loss of immune tolerance to pancreatic beta-cells leading to their destruction. Immune intervention therapies tested in T1D so far delayed progression but failed to restore tolerance, which partly explains their lack of durable clinical efficacy. RECENT FINDINGS: The role of beta-cells and islets themselves in dialogue with their micro- and macro-environment including the immune system and the intestinal microbiome is increasingly evident. Indeed, islets can both maintain and break immune tolerance. Some recent immune therapies in cancer that block immune regulation also break tolerance. Induction of immune tolerance requires activating immune activation too, whereas immune suppression precludes this process. Immunotherapy alone my not suffice without engaging islets to restore tolerance and preserve beta-cell function. SUMMARY: New insight into the role of islet tissue and its interaction with its environment in preserving or breaking tolerance has contributed to understand the development of islet autoimmunity and T1D. Knowing which factors in islets and the immune system contribute to maintaining, breaking, and restoring the balance in the immune system is critical to prevent initiation and reverse disease progression, and guides the design of novel tolerogenic strategies for durable therapeutic intervention and remission that target both the immune system and distressed islets.

Rationale for treating primary Sjögren's syndrome patients with an anti-CD6 monoclonal antibody (Itolizumab).

CD6 is a cell surface receptor expressed on the majority of T cells and a subset of B cells. When expressed, CD6 contributes to lymphocyte activation through its extracellular domain 1, while adhesion and cellular migration are related to the extracellular scavenger receptor cysteine-rich domain (SRCR-D)-3 of CD6. Itolizumab, clone T1h, is a newly developed humanized IgG1 monoclonal antibody that targets CD6 SRCR-D1 and blocks immune activation. Itolizumab has been proposed to be effective in autoimmune diseases such as rheumatoid arthritis, Sjögren's syndrome and multiple sclerosis. In Sjögren's syndrome, the utilization of itolizumab as therapeutic option is reinforced by our recent observation that ALCAM, the CD6 ligand, is overexpressed and that CD6-positive T and B cells are detected within salivary glands from Sjögren's syndrome patients. In this study, itolizumab-positive target cells were characterized within both peripheral blood and salivary glands in order to provide rational for anti-CD6 treatment in Sjögren's syndrome.

Racotumomab: an anti-idiotype vaccine related to N-glycolyl-containing gangliosides - preclinical and clinical data.

Neu-glycolyl (NeuGc)-containing gangliosides are attractive targets for immunotherapy with anti-idiotype mAbs, because these glycolipids are not normal components of the cytoplasmic membrane in humans, but their expression has been demonstrated in several human malignant tumors. Racotumomab is an anti-idiotype mAb specific to P3 mAb, an antibody which reacts to NeuGc-containing gangliosides, sulfatides, and other antigens expressed in tumors. Preparations containing racotumomab were able to induce a strong anti-metastatic effect in tumor-bearing mice. Different Phase I clinical trials have been conducted in patients with advanced melanoma, breast cancer, and lung cancer. The results of these clinical trials demonstrated the low toxicity and the high immunogenicity of this vaccine. The induced antibodies recognized and directly killed tumor cells expressing NeuGcGM3. A Phase II/III multicenter, controlled, randomized, double blind clinical trial was conducted to evaluate the effect of aluminum hydroxide-precipitated racotumomab vaccine in overall survival in patients with advanced non-small cell lung cancer. The clinical results of this study showed a significant clinical benefit in the patients who were treated with the anti-idiotype vaccine.

EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

Nimotuzumab, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, has been used extensively in many solid tumors and confers significant survival advantage. The antibody has limited skin toxicity and is generally well tolerated. Similar to other anti-EGFR therapies, patients may relapse a few months after treatment. In this study we show for the first time, the use of Nimotuzumab along with Sirolimus has synergistic effect on tumor inhibition as compared with the drugs used individually, in Nimotuzumab responsive and nonresponsive cell lines. In vitro studies prove that while Sirolimus (25 nmol/L) affects the signal downstream to mammalian target of rapamycin (mTOR), Nimotuzumab (83 nmol/L) downregulates pTYR, pMAPK and pSTAT3 by 40%, 20% and 30%, respectively. The combination, targeting these two different signaling hubs, may be associated with the synergistic inhibition observed. In vivo, the use of half human therapeutic equivalent doses for both the drugs substantially reduces tumors established in nude as well as severe combined immunodeficiency (SCID) mice by EGFR overexpressing A-431 cells. The drug combination reduces cell proliferation and the expression of signal transduction molecules. Treated tumors are better differentiated as compared with those established in the control mice. Tumor microarray demonstrates that Nimotuzumab and the combination groups segregate independently to the Sirolimus and the control treatment. The combination uniquely downregulated 55% of the altered tumor genes, extending beyond the typical pathways associated with Nimotuzumab and Sirolimus downstream pathways inhibition. These results would suggest that this nontoxic drug combination improves therapeutic benefit even in patients with low-EGFR expression and severely immunocompromised because of their current medication.

Safety, immunogenicity and preliminary efficacy of multiple-site vaccination with an Epidermal Growth Factor (EGF) based cancer vaccine in advanced non small cell lung cancer (NSCLC) patients.

The prognosis of patients with advanced non small cell lung (NSCLC) cancer remains dismal. Epidermal Growth Factor Receptor is over-expressed in many epithelial derived tumors and its role in the development and progression of NSCLC is widely documented. CimaVax-EGF is a therapeutic cancer vaccine composed by human recombinant Epidermal Growth Factor (EGF) conjugated to a carrier protein, P64K from Neisseria Meningitides. The vaccine is intended to induce antibodies against self EGF that would block EGF-EGFR interaction. CimaVax-EGF has been evaluated so far in more than 1000 advanced NSCLC patients, as second line therapy. Two separate studies were compared to assess the impact of high dose vaccination at multiple anatomic sites in terms of immunogenicity, safety and preliminary efficacy in stage IIIb/IV NSCLC patients. In both clinical trials, patients started vaccination 1 month after finishing first line chemotherapy. Vaccination at 4 sites with 2.4 mg of EGF (high dose) was very safe. The most frequent adverse events were grade 1 or 2 injection site reactions, fever, headache and vomiting. Patients had a trend toward higher antibody response. The percent of very good responders significantly augmented and there was a faster decrease of circulating EGF. All vaccinated patients and those classified as good responders immunized with high dose at 4 sites, had a large tendency to improved survival.

Aberrant expression of CD6 on B-cell subsets from patients with Sjögren's syndrome.

CD6 is one of a pair of related genes encoding CD5-associated receptors on all T cells and a subset of B cells. The current availability of "T1h", a humanized anti-CD6 monoclonal antibody for B cell-mediated autoimmune disorders revives analysis of the B-cell subset expression of CD6, particularly in primary Sjögren's syndrome (SS). Refined phenotype of B-lymphocytes peripheral blood (PB), bone marrow and tonsils revealed that the overlap between the expression of CD6 is less close to that of CD5 than currently acknowledged. In contrast to CD5, CD6 is absent on transitional B cells, while present on mature and memory B cells. Interestingly, the PB proportion of CD6(+) B cells is decreased in patients with primary SS, as opposed to those with rheumatoid arthritis. The reduction in primary SS does not result from the shedding of CD6 from the membrane of B cells, but from the lowering of memory B lymphocytes. It may result from the ability of CD6 to make transmigration of CD27(+) memory B cells into the salivary glands (SGs) easier. Consistent with this view is our finding that CD166 (one of the ligands for CD6) is highly expressed on epithelial cells of patients' SGs. This study is relevant in that the humanized T1h anti-CD6 becomes an alternative to anti-CD20 for treatment of primary SS.

Nimotuzumab with chemoradiation confers a survival advantage in treatment-naïve head and neck tumors over expressing EGFR.

Head and neck cancer associated with the chewing of betel preparations, including tobacco, is common to South East Asia. We report a Phase IIB study in which ninety-two treatment naïve patients with advanced squamous cell carcinoma received standard therapy with or without an anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody (Nimotuzumab). In pretreatment samples, the tissue expression of ErbB family proteins and downstream molecules, including their association with clinical response and survival. Marker expression in tumor adjacent sections was evaluated by immunohistochemistry. The clinical benefit of Nimotuzumab (200 mg/dose, once a week for six weeks) in combination with radiotherapy or chemoradiation was assessed with respect to EGFR expression and intensity. Two antibodies, which recognized independent epitopes, were used to assess EGFR expression levels by immunohistochemistry. EGFR detection using mR3, an antibody with similar specificity to Nimotuzumab, correlated significantly with the expression of ErbB3 (p<0.05), PCNA and pMAPK (p<0.001). Although EGFR expression showed a significant relationship to patient survival in patients treated with Nimotuzumab and chemoradiation (p=0.02), pMAPK expression did not (p=0.07). Interestingly, EGFR overexpression (as defined by mR3) correlated directly with overall survival in this group (p=0.01). This data supports a model of basal activation of the EGFR signal transduction pathway in these oropharyngeal tumors. Detection of EGFR by immunohistochemistry could define a subset of treatment naïve Head and Neck cancer patients who may benefit from receiving EGFR targeted therapies in combination with chemoradiation.

Construction of a recombinant non-mitogenic anti-human CD3 antibody.

IOR-T3, a mouse monoclonal antibody specific for human CD3, has been successfully used in the treatment of acute transplant rejection due to its potential as T-cell immunosuppressant. In the present work we report the construction of a human IgG1 chimeric variant of IOR-T3, named T3q. In order to reduce the T-cell activating capacity of the newly obtained chimeric molecule, the two leucine residues at positions 234 and 235 of the CH2 region were replaced by alanines, obtaining the T3q(Ala/Ala) molecule. In vitro evaluation of T3q and T3q(Ala/Ala) showed that there were no differences in the recognition of human CD3 in comparison with murine IOR-T3. However, the Fc-mutated version T3q(Ala/Ala) displayed a much weaker FcgammaR binding capacity than the unmutated chimeric molecule T3q, as well as a reduced ability to induce T-cell proliferation, proinflammatory cytokine release (TNFalpha and IL-6), and early activation surface marker expression (CD25 and CD69). We also found that, unlike treatment with T3q, the reduction in T-cell proliferation was less marked on CD8(+) cells compared to the CD4(+) cells after treatment with T3q(Ala/Ala). These properties make T3q(Ala/Ala) an attractive clinical alternative as an immunoregulatory agent endowed with reduced toxicity.

Rationale for Targeting CD6 as a Treatment for Autoimmune Diseases.

CD6 is a 105-130 kDa surface glycoprotein expressed on the majority of T cells and a subset of B cells. The human cd6 gene maps to chromosome 11, and the expression of its protein product is tightly regulated. CD6 mediates cellular adhesion migration across the endothelial and epithelial cells. In addition, it participates in the antigen presentation by B cells and the subsequent proliferation of T cells. CD6 may bind in trans to surface glycoproteins (such as ALCAM and 3A11), or to microbial lipopolysaccharides, and may bind in cis to endogenous ligands (such as CD3 and CD5), and thereby deliver a costimulatory signal. Transinteractions are reinforced during autoimmune diseases (e.g., rheumatoid arthritis (RA), Sjögren's syndrome, and multiple sclerosis) and some cancers. Based on experimental data and on clinical results in RA and psoriasis, we believe that the recent humanized anti-CD6-specific mAb T1h may act as a regulator of the immunological response in addition to its function as an anti-T- and -B cell agent.

Interferon-alpha conditioned sensitivity to an anti-epidermal growth factor receptor monoclonal antibody in a human lung cancer cell line with intermediate expression of the receptor.

Targeted therapy to the epidermal growth factor receptor (EGFR) seems to be related to its expression level on tumor cells. Interferon-alpha (IFN-alpha) induces growth inhibition but also may up-regulate the EGFR expression in some cancer cell lines. We aimed to determine whether the IFN-alpha combined with an EGFR-specific monoclonal antibody (nimotuzumab) may affect the growth of human tumor epithelial cell lines with different EGFR expression levels. H125, a lung adenosquamous carcinoma, and A431, a vulvar epidermoid carcinoma, cell lines express intermediate and high levels of EGFR, respectively, whereas MDA MB231, a breast adenocarcinoma cell line expresses undetectable levels of EGFR measured by flow cytometry/FACS. We found that IFN-alpha alone inhibited in a dose-dependent fashion the growth of all cell lines, but only up-regulated the EGFR expression in the lung carcinoma-derived cell line. Noteworthy, the combined treatment did not modify the complement-mediated cytotoxicity of the antibody although the antiproliferative activity of nimotuzumab in H125 cells in vitro increased when an IFN-alpha-conditioning treatment was used. In conclusion, this study may provide insights about the rational use of EGFR inhibitors into the immunopharmacological management of targeted therapies including the IFN-alpha for lung cancer.

Chemotherapy induced transient B-cell depletion boosts antibody-forming cells expansion driven by an epidermal growth factor-based cancer vaccine.

Cancer vaccines efficacy may improve inducing a rapid and persistent immune response, early at diagnosis along with standard therapies. EGF chemically conjugated to the carrier protein P64k from Neisseria meningitidis in montanide ISA 51 adjuvant is under evaluation, aiming to stimulate a B-cell response. High-dose cyclophosphamide and doxorubicin after priming enhanced the long-term frequency of EGF-specific antibody-forming cells (AFC) of IgM and IgG isotypes, but not the P64k response. Resulting combination, limitedly operational in Btk deficient xid mice, suggests that preferential B-cell lymphocyte space promoted by cyclophosphamide facilitates remaining EGF-specific AFC undergo homeostatic proliferation driven by boosting, amplifying the response.

Epidermal growth factor receptor modulates the tumorigenic potential of melanoma.

Potential contribution of the Epidermal Growth Factor Receptor (EGFR) in melanoma immunobiology remains unclear, in part due to a lack of experimental models. We demonstrated previously that B16F10 melanoma transfected with the full length cDNA of the human EGFR increases the tumor cell proliferation in vitro. To further study its contribution in vivo, EGFR-transfected B16F10 cells were inoculated in syngenic C57BL/6 mice and its tumorigenic capacity was compared with the parental melanoma. Contrary to the observed in vitro effect, EGFR-transfected B16F10 cells displayed a delayed tumor growth rate in vivo, correlating inversely to the transgene expression. Interestingly, resulting tumors showed a downregulation of the EGFR transgene expression. Contrastingly, parental and EGFR-transfected B16F10 cells exhibited a similar tumorigenic potential in immunocompromised subjects, persisting the EGFR transgene expression. These results document the adaptability of melanoma to growth in immunocompetent individuals. Moreover, the potential EGFR expression during the melanoma outgrowth that would be downregulated by interacting with the host immune system during the tumor evolution is not excluded and which may be dissected in this model.

Towards the definition of a chimpanzee and human conserved CD6 domain 1 epitope recognized by T1 monoclonal antibody.

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.

Priming and boosting determinants on the antibody response to an Epidermal Growth Factor-based cancer vaccine.

Epidermal Growth Factor chemically conjugated to P64k carrier protein from Neisseria meningitidis emulsified in Montanide ISA 51 adjuvant is a cancer vaccine under clinical evaluation. We explored the influence of priming and boosting variables on the antibody response in mice. An apparently low dose fractionated in multiple anatomical sites at priming accelerated the induction and enhanced the maximal antibody response, with a long-lasting effect. Moreover, shortening the boosting time reduces the antibody persistence. Repeatedly boosting shift subjects to good antibody-responders, maintaining the epitope immunodominance. We conclude that optimizing immunopharmacological determinants contribute to an earlier, stronger and prolonged anti-EGF antibody persistence.