Understanding how the immune system affects cancer development and progression remains one of the most challenging questions in immunology.
How can we most effectively inhibit cancer-induced immunosuppressive mechanisms at the tumor site to boost the host-protective anti-tumor effects of preexisting or therapeutically induced immunity without concomitantly inducing life-threatening autoimmunity? This is probably the most urging and important question in all of tumor immunology.
We are developing an innovative approach based on the use of single-domain antibodies and phage display to efficiently generate new molecules able to stimulate the main effector cells, i.e. NK cells through CD16 and NKGD2 and T cells through CD3, in a target cell-dependent way, within the tumor microenvironment.
Noninvasive early detection of breast cancer through the use of biomarkers is urgently needed since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery.
A crucial issue is the availability of relevant markers and corresponding antibodies to develop effective diagnostic but also therapeutic approaches. We have developed an original phage display selection procedure that allows the isolation of single domain antibodies (sdAbs) against new tumor markers. Using these new anti-tumor sdAbs, we are elaborating new innovative approaches for diagnosis and/or target discovery.
SdAbs are endowed with original features that make them particularly interesting for the generation of neutralizing antibodies.
Because they are constituted of a single immunoglobulin domain, these small antibody fragments (13 kDa) are capable of binding buried epitopes normally inaccessible to conventional antibodies. Moreover, sdAbs are extremely stable and remain functional in a reducing environment precluding the formation of disulfide bonds that are mandatory to conventional immunoglobulin domains. We are exploiting these properties to generate binders able to block functional and thus conserved epitopes of the Env protein of HIV-1 or to generate intracellular antibodies able to neutralize HIV-1 Nef regulatory protein in infected cells. Similar approaches using intracellular antibodies are being developed for cancer therapy.
We now appreciate that the immune system plays a dual role in cancer : it can not only suppress tumor growth by destroying cancer cells or inhibiting their outgrowth but also promote tumor progression either by selecting for tumor cells that are more fit to survive in an Darwinian-like process, or by establishing conditions within the tumor microenvironment that facilitate tumor outgrowth.
Tumor infiltration by IFN-\gamma producing Th1 CD4+ T cells and CD8+ T cells, and the presence of cytokines such as IFN-\gamma and TNF-\alpha that promote tumor control, have been associated with an improved prognosis for patients with many different cancers. Remarkably, the type and density of T cells infiltrating cancers was found to be a more powerful prognostic indicator than previous pathological criteria for tumor staging and was even more predictive than correlating disease progression with oncogene expression.
Similarly, several lines of evidence indicate that NK cells or their receptors have a role in immuno-surveillance of spontaneous tumors, including in humans. Indeed, some studies showed that low NK lytic activity is associated with cancer risk. However, progressing tumors have evolved mechanisms to escape NK cell control such as the shedding of soluble NKG2D ligands that function as decoys for the activating NKG2D receptor on NK cells. The tumor microenvironment of progressing tumor can also decrease the level of activating receptors and increase the expression of inhibitory receptors of infiltrated NK cells, leading to their functional impairment.
These observations have led to the concept of immunoedition stating that clinically invisible tumor cells are continuously eradicated by the immune systems (elimination). In rare cases, some tumor cells might survive, establishing a balance between growth and cell lysis (the equilibrium phase) leading to the selection of cells able to evade the immune system via the induction of an immunosuppressive state within the tumor microenvironment. These tumor cells may then enter the escape phase, leading to a clinically apparent disease.
This hostile microenvironment is generated by the tumorigenic process promoting a state of immunosuppression which is a barrier to efficient cell mediated immunity and immunotherapy. Our aim is thus to boost the state of activation of tumor infiltrated effector cells and/or to recruit new effector cells within the tumor microenvironment.
To do so, we are using an original format of recombinant bispecific antibody based on the use of llama-derived sdAbs fused in a linker free manner and in a proper orientation using the CH1/C\kappa domains of human IgG1 as heterodimerization motifs. These so-called bsFabs are extremely stable and efficiently produced in E. coli.
We are using bsFabs to polyclonally activate T cells in a MHC independent way through CD3, or to activate NK cells using different signaling receptors involved in their natural cytotoxicity (NKG2D) or in their antibody dependent mode of action (CD16).
SdAbs can efficiently be generated using immunization of llamas with various antigens including tumor cell line or human biopsies (see the nanobody platform page). Phage display libraries are efficiently built since their constructions do not necessitate a combinatorial step (unlike single chain Fv fragments).
We have generated several libraries using breast cancer biopsies. An innovative phage display procedure named Masked Selection has been designed to strongly favor the selection of tumor-specific or overexpressed markers using cell lysates (for diagnostic purposes) or intact tumor cells (for therapeutic targeting) during the selection procedures.
Several tumor specific sdAbs have been isolated and used to create multiplexed bead arrays allowing the sensitive detection and concentration determination of several tumor markers using low amounts of samples.
The identity of new tumor markers can be determined using immunoprecipitation combined to mass spectrometry studies.
These approaches are been applied to breast cancer but can be applied to various type of cancer.
Antibody mediated neutralization is a crucial means of host resistance to many pathogens. During HIV-1 infection, most antibodies generated by patients target the Env protein which is interacting with receptor CD4 and co-receptors CCR5 or CXCR4. To evade this immune response, HIV-1 has evolved many strategies including high sequence variability, protection of sensitive epitopes by a shield of carbohydrate moieties, as well as conformational and entropic masking. Consequently, the neutralizing antibody response during HIV-1 infection is weak and narrow, and only a few monoclonal antibodies with broad neutralization breadth have been isolated so far.
Despite this complexity, the HIV-1 Env protein must retain conserved determinants that mediate CD4 and CCR5 binding. Unfortunately, these epitopes are buried, protected by a dense glycosylation and are not easily targeted by conventional antibodies.
Due to their small size, llama-derived single domain antibodies (sdAbs) can bind to epitopes usually not accessible to classical antibodies. We are using this property to generate neutralizing antibody fragments targeting the CD4 or co-receptor binding sites of HIV-1 Env protein. Various selection strategies are performed to isolate panels of single-domain antibodies with broad neutralization properties.
Because sdAbs are also characterized by an exceptional stability, they remain functional when produced in the reducing environment of eukaryotic cells. Owing to these properties, sdAbs can behave as intracellular antibodies (intrabodies) able to neutralize their target in living cells.
We have used this original feature to generate an intrabody targeting the HIV-1 Nef, a protein expressed early in the infection which protect the infected cell from attack by cytotoxic T cells and increase virus infectivity. This sdAb is able to inhibit most of the critical activities of both in vivo and in vitro in CD4-positive T lymphocytes, including CD4 downregulation, infectivity enhancement, and thymic CD4+ T-cell maturation defects.
We have extended the function of this sdAb by fusing a SH3 domain able to target another Nef epitope. The resulting molecule named Neffin is able to block all functions of Nef, including MHC-1 downregulation and inhibitory activity on phagocytosis. This construct can represent an efficient tool to develop new antiviral strategies targeting Nef.
From left to right: Elise, Rachid, Daniel, Patrick, Benjamin, Damien, Marc, Klervi, Lauren, Stéphane, Brigitte, Corinne