Biotechnology and Immunotherapy Notes

Biotechnology & Immunotherapy

Biotechnology

• Using organisms (or their products) as tools.

• Produce pharmaceuticals and commercial products.

• Enhance biological research.

• Clean environment.

• New forensic techniques.

Uses for Antibodies (Abs) in Biotechnology

• Specificity for individual compounds makes Abs optimal for:

  • Assays

    • Qualitative (identify)

    • Quantitative (# of)

  • Selection/Isolation of a product

Monoclonal Antibodies (mAbs)

• "One clone"

• Abs made from one B cell clone line.

• Clonal expansion.

• One type of Ab made against one epitope on an Antigen (Ag).

Polyclonal Antibodies

• "Many clones"

• Abs made from different B cells (different clone lines).

• Different Abs target different epitopes on the same Ag.

Producing Monoclonal Abs

1. Mouse challenged with Ag.

2. Spleen Cells are harvested.

3. Fusion with Myeloma Cells to create Hybridomas.

4. Culture in HAT Medium.

5. Select for positive cells.

6. Harvest monoclonal antibodies.

Producing Polyclonal Abs

1. Inject Ag into rabbit.

2. Ag activates B cells.

3. Plasma B cells produce polyclonal antibodies.

4. Obtain antiserum from rabbit containing polyclonal antibodies.

Monoclonal vs. Polyclonal Abs

ELISA (Enzyme-Linked Immunosorbent Assay)

• Abs (mono/poly) against epitope.

• Abs linked with fluorescent enzymes.

• If Ag present, samples fluoresce.

• Greater fluorescence = more Ag present.

Primary, Secondary & Capture Abs in ELISA

• Primary = Ab that binds to Ag (may or may not be labelled w/enzyme).

• Secondary = Ab that binds to primary Ab (always labelled w/enzyme).

• Capture = Ab in wells that fastens Ag (never labelled w/enzyme).

4 Types of ELISA

• Direct: One anti-Ag Ab used (primary).

• Indirect: One anti-Ag and one Anti-Ab Ab used (primary & secondary).

• Sandwich: Ag sandwiched between two Anti-Ag Abs (capture & primary).

• Subtypes: Direct & Indirect variants exist for Sandwich ELISA.

Direct ELISA

1. Add Ag to wells.

2. Add primary Ab linked with fluorescent enzyme to wells.

3. Wash with buffer.

4. Add substrate for enzyme.

5. Observe fluorescence intensity.

Indirect ELISA

1. Add Ag to wells.

2. Primary Ab added to wells.

3. Wash with buffer.

4. Add secondary Ab linked to fluorescent enzyme to wells.

5. Wash with buffer.

6. Add substrate for enzyme.

7. Observe fluorescence intensity.

Direct vs. Indirect ELISA

Direct/Indirect Sandwich ELISA

1. Fix capture Ab to wells.

2. Add Ag to wells.

3. Add primary Ab to wells (direct: enzyme-linked; indirect: not).

4. Wash with buffer.

5. If indirect variant, add secondary Ab to wells.

6. Wash with buffer (indirect).

7. Add substrate for enzyme to wells.

8. Observe fluorescence intensity.

Direct/Indirect Sandwich Advantages/Disadvantages

• Advantages

  • Much more sensitive (2-5x)

  • Much more specific

• Disadvantages

  • Hardest

  • Possible cross-reactivity between capture and other Abs

Direct Sandwich ELISA: At-Home HCG Pregnancy Tests

• Example provided with description of how the test works.

Flow Cytometry

• Machine that can efficiently count, characterize and sort individual cells (or even proteins) based on light refraction and labels.

• Can measure physical characteristics (size, shape, etc.) of cells.

• Abs (linked with an fluorescent molecule like FITC) are often used to label cells.

• Fluorescence-activated cell sorting (FACS).

Flow Cytometry Applications

• Used in clinical trials and research for reasons including

  • Apoptosis rates

  • Tumor cell quantity & type

  • HIV research

  • WBC, RBC and platelet count, development and characteristics

  • Soluble Ags

  • Reverse ELISA

Immunoaffinity Chromatography

• Isolating a specific protein product

• Add Abs matching desired protein to column.

• Add protein solution.

• Desired proteins bind to Abs and other substances wash away.

• Chemically separate protein from Ab and voila!

Biotechnology and Immunological Research

• Abs can be isolated using chromatography for study, ELISA, etc.

• Abs can be altered to make new medicines.

Antibody Purification

• Isolating desired Abs

• Ag coated beads (conjugates)

• Add Ab mixture

• Matching Abs bind to conjugate

• Column too small for immune complex and other Abs are washed away.

• Chemically separate Abs from Ags and voila!

Immunotherapy

• Medical regulation of immune system

  • Upregulate (activate, boost, etc.)

    • Ex. Vaccinations

  • Downregulate (suppress)

    • Ex. Glucocorticoids

• Can use apheresis, cytokines, Ags, synthetic compounds or cells

• Frontier:

  • Cancer therapies

  • Hypersensitivity therapies

Immunoadsorption

• Removal of autoimmune Abs from bloodstream.

• Very specific and more efficient than plasmapheresis (membrane-based separation).

• Extracorporeally filter patient’s blood through an immunoaffinity chromatography column.

• Use ligands that target only autoantibodies.

Engineered Abs

• Abs can be modified to generate new therapies.

• Abs that target receptors or neutralize substances to restore homeostasis

  • Ex. Catumaxomab as a cancer therapy

    • Chimeric Ab (fusion of two different Abs)

    • Bi-specific, tri-functional

Catumaxomab Rationale

• Bispecific

  • Recognizes EpCAM (cancer Ag) and CD3 (T cells)

• Tri-functional

  • Attach to tumor cell

  • Attach to T cell

  • Attach to leukocytes with corresponding Fc receptors

Malignant Ascites (MA)

• Abdominal cancer that causes edema (blocked flow of lymph, etc.)

• Solid tumor

• European Union approved catumaxomab for MA when standard therapy is not available/effective

Catumaxomab Challenges

• Human anti-mouse and human anti-rat Abs can dampen efficacy

• Cytokine release syndrome (CRS)

  • Pyrexia, vomiting, abdominal pain

Catumaxomab Marketing Withdrawal

• Relatively effective and safe (side-effects generally manageable) but…

• Company discontinued in EU because of bankruptcy

Adoptive Cell Therapy (ACT)

• Filter out leukocytes (apheresis)

• Autologous or allogous

• Can enhance and/or grow them

• Send them to battle!

CAR-T Therapy

• Chimeric Ag Receptor (CAR) T cell

• CAR recognizes cancer Ag independent of MHC

• Cancer Ag = DAMP example

• Adoptive T cell therapy

CAR-T Therapy Strategy

1. Apheresis & Purification

2. Genetic modification of cancer-specific Ag receptor

3. Proliferation

4. Transplantation

Apheresis & Purification (CAR-T)

• Filter out blood for leukocytes

• Purify T cells (both CD4+ and CD8+) with immunoaffinity chromatography (or comparable method)

Genetic Engineering of CAR-T Cells: Transduction

• Add CAR gene via a defective retroviral vector

  • Viral reproductive sequences not in reverse transcribed segment

  • Virion must exhibit tropism for both CD4 and CD8 T cells

• Simplified process:

  1. Infect a “packaging cell” w/modified virus.

     • Genes of interest (CAR)

     • Viral reproductive sequences separated (in packaging cell but not its DNA)

  2. Packaging cell releases virions that cannot reproduce (missing viral reproductive sequences).

  3. Host cell receives and only expresses genes of interest

     • No virions produced

Proliferation

• Several possible methods (can use multiple)

  • Proliferative cytokines, especially IL-2

  • Activate co-stimulatory factor CD28 via monoclonal Ab beads

  • Activate CD3 via monocolonal Ab beads

  • Autologous or artificial APCs loaded w/Ag

CAR Structure

• Contains Ab-derived protein segment

  • Single chain variable Fragment (scFv)

• Signal transduction protein segments

  • Three domains:

    • Ectodomain

    • Transmembrane domain

    • Endodomain

Ectodomain

• Outside of membrane

• scFv = VH, VL & linker

  • VH & VL = paratope

    • Recognize cancer Ag (epitope)

  • Linker connects and arranges VH & VL (need to be side by side)

  • Spacer connects to transmembrane domain

• Signal peptide sends CAR to RER for protein secretion pathway

  • Removed as CAR enters RER

Transmembrane Domain

• Bridge between outside and inside cell (ecto and endodomains respectively)

• Once scFv activated, transduces signal to endodomain

Endodomain

• Combination of several signaling proteins

• Transduce multiple signals simultaneously

  • Cytotoxicity

  • Proliferation

  • Cytokine Production

  • Survival

• ζ chain (also called CD3 ζ chain) → activation & cytotoxicity

• CD28 tail → IL-2 (activation, proliferation & survival)

• CD137 tail → Augments CD28 tail’s effects & survival

CAR Generations

• 1G: Contains only the chain

• 2G: Contains one co-stimulatory domain (4-1BB or CD28) with the chain

• 3G: Contains two co-stimulatory domains. (4-1BB or CD28) with the chain

CAR-Mediated Cytotoxicity

• The scFV binds to the tumor associated antigen, CD19, which sends a signal to the T-cell

• The T-cell is activated to kill the tumor cell

• Perforin and granzyme are released from the activated T-cell via exocytosis Perforin

• Pores are created within the tumor cell membrane, allowing granzyme to kill the tumor cell

Current Applicability

• Approved for B cell cancers

  • Pediatric ALL (leukemia type), several non-Hodgkin’s lymphomas, multiple myeloma (MM)

• Clinical trials on solid tumors (including, melanoma, ovarian, pancreatic, lung)

Efficacy & Advantages

• Efficacy: 70-90+% (even in relapsed patients) in ALL

  • Relatively most effective against treatment-resistant cancers and when other treatments are failing

• Efficacy: 93% in remission in MM study

  • No other effective treatments available for MM

  • Best when chemotherapy given just before CAR-T administered

• Advantages: highly effective, short treatment period (2 weeks vs months of chemo), and protection from relapses (memory!)

Main Side-Effects & Costs

• Cytokine release syndrome (CRS) → cytokine storm

  • Data ex. 10/133 experienced serious CRS

  • Counteract with steroids and IL-6 inhibitor

• Neurotoxicity (confusion, headaches, seizures, loss of speech, coma)

  • Data ex. 7/133 experienced life-threatening neurotoxicity in one study

• Hypogammaglobulinemia due to B cell decline (CD19 expressed by normal B cells too [to a lesser degree]).

• Price: $373,000$ (best price) alone

  • With hospitalization, etc. estimated 1.5 million per patient

Armored CAR-T/TRUCK Therapy

• CAR-T cells redirected for universal cytokine killing (TRUCK)

• Confer IL-12 production and constitutive CD40L expression on T cells

• Likely much more effective at combatting solid tumors

• IL-12 encourages IFN-γ production by T & NK cells, stimulating innate immunity (mφ, NK, iNKT), Tc and Th1 cells

  • Prevents immunosuppression

  • IFN-γ production boosts IL-12 production by mφ

    • + feedback

• CD40L (primed) T cells exhibit greater cytotoxicity.

SMDC Car-T Therapy

• Small molecule drug conjugate (SMDC) acts as bridge between CAR-T cells and tumor.

• Cancer cells overexpress folate receptor.

  • Great Ag target

  • Increase specificity = increase safety

• Create conjugate of folate and FITC

  • Folate receptor binds to folate.

  • FITC is fluorescent molecule (flow cytometry dye) that can be target of scFv

• Once CAR-T cell and tumor cell are bridged, T cell is activated and kills tumor cell.

TCR Therapy

• Genetically engineer T cells to target specific cancer peptides (DAMP) presented on MHC class I

• Adoptive cell therapy

• In clinical trials for solid tumors

Helminth Immunotherapy

• Helminths can be utilized to eliminate hypersensitivities, especially autoimmunity

• Also requires balance with microbiota

• Goal: restore homeostasis

Helminth Survival

• Various means to stay alive in host including motility, variant surface glycoprotein (VSG) and immunomodulation

  • Motility: catch me if you can!

Helminth Immunomodulation

• Elicit Th2 response

  • IgE, eosinophils and so on

• Modulate Th1 response

• Elevate Treg response

  • IL-10 and TGF-β

• Induce leukocyte apoptosis

• Degrade C3 complement protein

• Neutralize ROS attacks

Helminth Evolutionary Influence on Immune System

• Jawed vertebrate evolution parallels helminth diversification

• γδ T cells prevalent in gut epithelium

• Most selective pressure on gut-associated immunological genes

Old “Frienemies”

• Human immune system coevolved with helminth occupation

• Helminth removal disrupts or destabilizes immune system

  • Autoimmunity and allergies

• Rectify with safe* introduction of helminths or their products

Gut Microbiota

• Bacteria and fungi that inhabit intestines

• Mutualistic relationship with host

  • Provide Vitamin K and short-chain fatty acids

  • Inhibit pathogenesis

  • Receive nutrients and habitat in return

• Risks

  • Systemic shock if breach intestinal barriers

  • Dysbiosis (pro-inflammatory increase in community)

Containment Approach

• High diversity and appropriate composition is healthy

• Dysbiosis can lead to immunopathology

  • Disproportionate # of pro-inflammatory bacteria

  • Inflammatory bowel disease (IBD)

• Best to tolerate and regulate microbiota presence

  • Monitor microbiota Ags, produce IgA, IL-22, etc.

Microbiota & Helminths Interact

• Some microbiota can attack helminths and vice versa

• Some [opportunistic] microbiota are helped by helminths and vice versa

• But they can get along

  • Macaque IBD and dysbiosis corrected with helminth

Triumverate

• Host, Microbiota, Parasite

• Understanding interplay between host (us), microbiota and parasites is key to addressing hypersensitivities